JP2005506033A - Prostate cancer diagnostic method, prostate cancer modulator screening composition and method - Google Patents

Abstract

Described herein are genes whose expression is upregulated or downregulated in prostate cancer. Furthermore, genes whose expression is upregulated or downregulated in drug resistant prostate cancer cells have been described. Related methods and compositions that can be used in the diagnosis and treatment of prostate cancer have been identified. Further described herein are methods that can be used to identify modulators of prostate cancer.

Description

前記１４μｌ混合液を７０℃で１０分間インキュベーションし、その後氷上に置いた。
【０３５０】
逆転写法には、下記の混合液を用いた：

前記溶液を、ハイブリダイゼーション反応液に加え、４２℃で３０分間インキュベーションした。その後、ＳＳＩＩ １μｌを添加し、更に１時間インキュベーションし、その後氷上に置いた。
【０３５１】
５０ｘｄＮＴＰ混合液は、２５ｍＭの冷ｄＡＴＰ、ｄＣＴＰ、及びｄＧＴＰ、１０ｍＭ ｄＴＴＰを含有し、及び１００ｍＭ ｄＡＴＰ、ｄＣＴＰ、及びｄＧＴＰを各々２５μｌ；１００ｍＭ ｄＴＴＰ １０μｌを、Ｈ_２Ｏ １５μｌに添加することにより作成した。
【０３５２】
ＲＮＡ分解を以下のように行った。８６μｌのＨ_２Ｏに、１．５μｌ １Ｍ ＮａＯＨ／２ｍＭ ＥＤＴＡを添加し、６５℃で１０分間インキュベーションした。Ｕ−Ｃｏｎ ３０については、５００μｌ ＴＥ／試料を、７０００ｇで１０分間回転し、精製のためにフロースルーを使用した。キアゲン精製については、ｕ−ｃｏｎ回収した材料を、５００μｌの緩衝液ＰＢ中に懸濁し、Ｑｉａｇｅｎプロトコールを用いて進めた。ＤＮＡｓｅ消化については、ＤＮＡｓｅ／３０μｌ Ｒｘの１／１００希釈物を１μｌ添加し、３７℃で１５分間インキュベーションした。９５℃で５分間インキュベーションし、ＤＮＡｓｅを変性した。
【０３５３】
試料調製
試料調製のために、Ｃｏｔ−１ ＤＮＡ、１０μｌ；５０ｘ ｄＮＴＰ、１μｌ；２０ｘ ＳＳＣ、２．３μｌ；ピロリン酸ナトリウム、７．５μｌ；１０ｍｇ／ｍｌニシン精子ＤＮＡ；１／１０希釈物１μｌを添加し、最終容量２１．８とした。高速減圧下で乾燥した。Ｈ_２Ｏ １５μｌ中に再懸濁した。１０％ＳＤＳ ０．３８μｌを添加した。９５℃で２分間加熱し、２０分間かけて緩徐に室温まで冷却した。スライド上に置き、６４℃で一晩ハイブリダイズした。ハイブリダイゼーション後洗浄した：３ｘＳＳＣ／０．０３％ＳＤＳ：２分間、Ｈ_２Ｏ ２５０ｍｌ中２０ｘＳＳＣ ３７．５ｍｌ＋１０％ＳＤＳ ０．７５ｍｌ；１ｘＳＳＣ：５分間、Ｈ_２Ｏ ２５０ｍｌ中２０ｘＳＳＣ １２．５ｍｌ；０．２ｘＳＳＣ：５分間、Ｈ_２Ｏ ２５０ｍｌ中２０ｘＳＳＣ ２．５ｍｌ。スライドを乾燥し、適当なＰＭＴ及びチャネルで走査した。
【０３５４】
実施例２：ヒト前立腺癌のタキソール抵抗性異種移植片モデル
パクリタキセル（タキソール；Ｂｒｉｓｔｏｌ−Ｍｙｅｒｓ Ｓｑｕｉｂｂ社、プリンストン、ＮＪ）を含む治療投薬管理は、ホルモン治療抵抗性前立腺癌の臨床試験第ＩＩ相の治療において特に成功している（Ｓｍｉｔｈら、Ｓｅｍｉｎ． Ｏｎｃｏｌ．、２６（１ Ｓｕｐｐｌ ２）：１０９−１１（１９９９））。しかし、多くの患者が、最初に又は後にタキソール抵抗性となる腫瘍を発生している。タキソールに対する抵抗性に関連した遺伝子、又はタキソール抵抗性に対する反応を調節し、その結果治療に使用することができる遺伝子、又は患者におけるタキソール抵抗性を同定するために、下記の実験を行った。
【０３５５】
アンドロゲン非依存型ヒト細胞株ＣＷＲ２２Ｒを、ヌードマウスにおいて異種移植片として増殖した（Ｎａｇａｂｈｕｓｈａｎら、Ｃａｎｃｅｒ Ｒｅｓ．、５６（１３）：３０４２−３０４６（１９９６）；Ａｇｕｓら、Ｊ． Ｎａｔｌ． Ｃａｎｃｅｒ Ｉｎｓｔ．、９１（２１）：１８６９−１８７６（１９９９）；Ｂｕｂｅｎｄｏｒｆら、Ｊ． Ｎａｔｌ． Ｃａｎｃｅｒ Ｉｎｓｔ．、９１（２０）：１７５８−１７６４（１９９９））。最初に、これらの異種移植片腫瘍は、治療用量のタキソールに対し感受性であった。このマウスを、治療用量を下回る量で連続処置し、その腫瘍を３〜４週間成長させ、その後外科的に腫瘍を摘出した。その後個々のマウスからの腫瘍を細断し、小さい部分を健常なヌードマウスに注射し、腫瘍の第二継代を確立した。次にこのマウスを同じ治療用量を下回る量のタキソールで連続処置した。この過程を１４回反復し、異種移植片腫瘍の各世代の一部を収集した。各世代で、治療用量のタキソールに対する抵抗性は増加した。この過程の最後までに、腫瘍は治療用量のタキソールに対し完全に抵抗性となった。次に各世代の腫瘍から得たＲＮＡを単離し、個々のｍＲＮＡ種を、およそ３５，０００種の独自のｍＲＮＡ転写物を問い合わせるためにプローブで、特注のＡｆｆｙｍｅｔｒｉｘ ＧｅｎｅＣｈｉｐ（登録商標）オリゴヌクレオチドマイクロアレイを用いて定量した。漸増するタキソール−抵抗性腫瘍の引き続きの継代期間において、統計学的に有意な上方制御、又は下方制御を示した遺伝子を、選択した。ただひとつの遺伝子が、有意に上方制御されたのに対し、２４種の遺伝子が下方制御された。これらを、表１０に示す。
【０３５６】
前立腺癌において過剰発現されていることが同定された遺伝子配列を使用し、公開のＤＮＡデータベースからコード領域を同定した。これらの配列は、公知のタンパク質をコードしている遺伝子の同定に使用するか、又はこれらは、ＦＧＥＮＥＳＨ（Ｓａｌａｍｏｖ及びＳｏｌｏｖｙｅｖ、Ｇｅｎｏｍｅ Ｒｅｓ．、１０：５１６−５２２（２０００））のようなエキソン推定アルゴリズムを用い、ゲノムＤＮＡからのコード領域の推定に使用するかのいずれかであった。加えて、当業者には、表１〜１６に提供された典型的アクセッション番号に従い、いかにしてｕｎｉｇｅｎｅクラスタの同定及び配列情報を得るかが理解されると思われる（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／ＵｎｉＧｅｎｅ／）。
【０３５７】
【表１】発現配列タグを含み、Ａｆｆｙｍｅｔｒｉｘ／Ｅｏｓ Ｈｕ０１ ＧｅｎｅＣｈｉｐアレイを用いて分析した時、前立腺腫瘍組織において正常組織と比べ示差的に発現された遺伝子を示す。遺伝子の最高発現を示している前立腺腫瘍試料及び様々な正常組織試料において発現された各遺伝子の相対量を示す。

【０３５８】
【表１Ａ】表１のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーの寄託番号を示す。本発明者らは、各プローブセットについて、そこからオリゴヌクレオチドをデザインした遺伝子クラスタ番号を列記した。遺伝子クラスタは、Ｇｅｎｂａｎｋ ＥＳＴ及びｍＲＮＡ由来の配列を用いて集計した。これらの配列は、クラスタリングアンドアライメントツール（Ｃｌｕｓｔｅｒｉｎｇ ａｎｄ Ａｌｉｇｎｍｅｎｔ Ｔｏｏｌｓ、ＤｏｕｂｌｅＴｗｉｓｔ社、オークランド、ＣＡ）を用い、配列類似性を基にクラスタ化した。各クラスタを含む配列のＧｅｎｂａｎｋ寄託番号は、「Ａｃｃｅｓｓｉｏｎ」欄に列記した。

【０３５９】
【表２】前立腺腫瘍組織において正常前立腺組織と比べ示差的に発現された表１に示した遺伝子の寄託番号の好ましいサブセットを示す。

【０３６０】
【表２Ａ】表２のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーの寄託番号を示す。本発明者らは、各プローブセットについて、そこからオリゴヌクレオチドをデザインした遺伝子クラスタ番号を列記した。遺伝子クラスタは、Ｇｅｎｂａｎｋ ＥＳＴ及びｍＲＮＡ由来の配列を用いて集計した。これらの配列は、クラスタリングアンドアライメントツール（Ｃｌｕｓｔｅｒｉｎｇ ａｎｄ Ａｌｉｇｎｍｅｎｔ Ｔｏｏｌｓ、ＤｏｕｂｌｅＴｗｉｓｔ社、オークランド、ＣＡ）を用い、配列類似性を基にクラスタ化した。各クラスタを含む配列のＧｅｎｂａｎｋ寄託番号は、「Ａｃｃｅｓｓｉｏｎ」欄に列記した。

【０３６１】
【表３】発現配列タグを含み、Ａｆｆｙｍｅｔｒｉｘ／Ｅｏｓ Ｈｕ０２ ＧｅｎｅＣｈｉｐアレイを用いて分析した時、前立腺腫瘍組織において正常組織と比べ示差的に発現された遺伝子を示す。遺伝子の最高発現を示している前立腺腫瘍試料及び様々な正常組織試料において発現された各遺伝子の相対量を示す。

【０３６２】
【表３Ａ】表３のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーの寄託番号を示す。本発明者らは、各プローブセットについて、そこからオリゴヌクレオチドをデザインした遺伝子クラスタ番号を列記した。遺伝子クラスタは、Ｇｅｎｂａｎｋ ＥＳＴ及びｍＲＮＡ由来の配列を用いて集計した。これらの配列は、クラスタリングアンドアライメントツール（Ｃｌｕｓｔｅｒｉｎｇ ａｎｄ Ａｌｉｇｎｍｅｎｔ Ｔｏｏｌｓ、ＤｏｕｂｌｅＴｗｉｓｔ社、オークランド、ＣＡ）を用い、配列類似性を基にクラスタ化した。各クラスタを含む配列のＧｅｎｂａｎｋ寄託番号は、「Ａｃｃｅｓｓｉｏｎ」欄に列記した。

【０３６３】
【表３Ｂ】ｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーのゲノム位置及び表３の寄託番号を示す。各々推定されたエキソンについて、本発明者らは、推定に使用したゲノム配列の給源を列記した。各推定されたエキソンのヌクレオチド位置も列記した。

【０３６４】
【表４】前立腺腫瘍組織において正常前立腺組織と比べ示差的に発現されている、表３において認められた遺伝子の寄託番号の好ましいサブセットを示す。

【０３６５】
【表４Ａ】表４のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーの寄託番号を示す。本発明者らは、各プローブセットについて、そこからオリゴヌクレオチドをデザインした遺伝子クラスタ番号を列記した。遺伝子クラスタは、Ｇｅｎｂａｎｋ ＥＳＴ及びｍＲＮＡ由来の配列を用いて集計した。これらの配列は、クラスタリングアンドアライメントツール（Ｃｌｕｓｔｅｒｉｎｇ ａｎｄ Ａｌｉｇｎｍｅｎｔ Ｔｏｏｌｓ、ＤｏｕｂｌｅＴｗｉｓｔ社、オークランド、ＣＡ）を用い、配列類似性を基にクラスタ化した。各クラスタを含む配列のＧｅｎｂａｎｋ寄託番号は、「Ａｃｃｅｓｓｉｏｎ」欄に列記した。

【０３６６】
【表４Ｂ】表４のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーのゲノム位置及び寄託番号を示す。各々推定されたエキソンについて、本発明者らは、推定に使用したゲノム配列の給源を列記した。各推定されたエキソンのヌクレオチド位置も列記した。

【０３６７】
【表５】前立腺癌において正常成人組織と比べアップレギュレーションされた１１７０種の遺伝子
表５は、前立腺癌において正常成人組織と比べアップレギュレーションされた１１７０種の遺伝子を示している。これらは、Ａｆｆｙｍｅｔｒｉｘ／Ｅｏｓ Ｈｕ０３ ＧｅｎｅＣｈｉｐアレイ上の５９６８０のプローブセットから選択し、「平均」前立腺癌対「平均」正常成人組織の比は、３．４４より大きいか又は等しかった。「平均」前立腺癌レベルは、７３種の前立腺癌中の８５ｔｈパーセンタイル値に設定した。「平均」正常成人組織レベルは、１６２種の非悪性組織の中の８５ｔｈパーセンタイル値に設定した。非特異的ハイブリダイゼーションの遺伝子特異的バックグラウンドレベルを取除くために、１６２種の非悪性組織の中の７．５ｔｈパーセンタイル値を、分子及び分母の両方から減じ、その後この比について評価した。

【０３６８】
【表５Ａ】表５、６及び７のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーの寄託番号を示す。本発明者らは、各プローブセットについて、そこからオリゴヌクレオチドをデザインした遺伝子クラスタ番号を列記した。遺伝子クラスタは、Ｇｅｎｂａｎｋ ＥＳＴ及びｍＲＮＡ由来の配列を用いて集計した。これらの配列は、クラスタリングアンドアライメントツール（Ｃｌｕｓｔｅｒｉｎｇ ａｎｄ Ａｌｉｇｎｍｅｎｔ Ｔｏｏｌｓ、ＤｏｕｂｌｅＴｗｉｓｔ社、オークランド、ＣＡ）を用い、配列類似性を基にクラスタ化した。各クラスタを含む配列のＧｅｎｂａｎｋ寄託番号は、「Ａｃｃｅｓｓｉｏｎ」欄に列記した。

【０３６９】
【表５Ｂ】表５、６及び７のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーのゲノム位置及び寄託番号を示す。各々推定されたエキソンについて、本発明者らは、推定に使用したゲノム配列の給源を列記した。各推定されたエキソンのヌクレオチド位置も列記した。

【０３７０】
【表６】前立腺癌において正常成人組織と比べアップレギュレーションされた細胞外又は細胞表面タンパク質をコードしている２８６種の遺伝子
表６は、恐らく細胞外又は細胞表面タンパク質である、前立腺癌において正常成人組織と比べアップレギュレーションされた２８６種の遺伝子を示している。これらは、表５についてのように選択され、及び推定されたタンパク質は、細胞外局在の指標である構造ドメイン（例えば、ｅｇｆ、７ｔｍドメイン）を含んだ。

【０３７１】
【表７】前立腺癌において正常成人組織と比べアップレギュレーションされた小分子標的をコードしている４２種の遺伝子
表７は、恐らく小分子標的である、前立腺癌において正常成人組織と比べアップレギュレーションされた４２種の遺伝子を示している。これらは、表５についてのように選択され、及び推定されたタンパク質には、薬物となりうる（ｄｒｕｇａｂｌｅ）構造の指標である構造ドメイン（例えば、プロテアーゼ、キナーゼ、ホスファターゼ受容体）が含まれた。機能ドメインは各遺伝子について示されている。

【０３７２】
【表８】前立腺癌において正常成人組織と比べ有意にダウンレギュレーションされた１３６種の遺伝子
表８は、前立腺癌において正常成人組織と比べ有意にダウンレギュレーションされた１３６種の遺伝子を示している。これらは、Ａｆｆｙｍｅｔｒｉｘ／Ｅｏｓ Ｈｕ０３ ＧｅｎｅＣｈｉｐアレイ上の５９６８０のプローブセットから選択し、「平均」正常前立腺対「平均」前立腺癌組織の比は、２より大きいか又は等しかった。「平均」正常前立腺レベルは、４種の正常前立腺組織の平均に設定した。「平均」前立腺癌レベルは、７３種の前立腺癌中の８５ｔｈパーセンタイル値に設定した。非特異的ハイブリダイゼーションの遺伝子特異的バックグラウンドレベルを取除くために、全ての組織の中の１０ｔｈパーセンタイル値を、分子及び分母の両方から減じ、その後この比について評価した。

【０３７３】
【表８Ａ】表８のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーの寄託番号を示す。本発明者らは、各プローブセットについて、そこからオリゴヌクレオチドをデザインした遺伝子クラスタ番号を列記した。遺伝子クラスタは、Ｇｅｎｂａｎｋ ＥＳＴ及びｍＲＮＡ由来の配列を用いて集計した。これらの配列は、クラスタリングアンドアライメントツール（Ｃｌｕｓｔｅｒｉｎｇ ａｎｄ Ａｌｉｇｎｍｅｎｔ Ｔｏｏｌｓ、ＤｏｕｂｌｅＴｗｉｓｔ社、オークランド、ＣＡ）を用い、配列類似性を基にクラスタ化した。各クラスタを含む配列のＧｅｎｂａｎｋ寄託番号は、「Ａｃｃｅｓｓｉｏｎ」欄に列記した。

【０３７４】
【表８Ｂ】表８のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーのゲノム位置及び寄託番号を示す。各々推定されたエキソンについて、本発明者らは、推定に使用したゲノム配列の給源を列記した。各推定されたエキソンのヌクレオチド位置も列記した。

【０３７５】
【表９】正常前立腺において前立腺癌と比べ有意にアップレギュレーションされた１００１種の遺伝子
表９は、前立腺癌において正常前立腺と比べ有意にアップレギュレーションされた１００１種の遺伝子を示している。これらは、Ａｆｆｙｍｅｔｒｉｘ／Ｅｏｓ Ｈｕ０３ ＧｅｎｅＣｈｉｐアレイ上の５９６８０のプローブセットから選択し、「平均」正常前立腺対「平均」前立腺癌組織の比は、８．１４より大きいか又は等しかった。「平均」正常前立腺レベルは、４種の正常前立腺組織の平均に設定した。「平均」前立腺癌レベルは、７３種の腫瘍試料中の８５ｔｈパーセンタイル値に設定した。非特異的ハイブリダイゼーションの遺伝子特異的バックグラウンドレベルを取除くために、全ての組織の中の１０ｔｈパーセンタイル値を、分子及び分母の両方から減じ、その後この比について評価した。

【０３７６】
【表９Ａ】表９のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーの寄託番号を示す。本発明者らは、各プローブセットについて、そこからオリゴヌクレオチドをデザインした遺伝子クラスタ番号を列記した。遺伝子クラスタは、Ｇｅｎｂａｎｋ ＥＳＴ及びｍＲＮＡ由来の配列を用いて集計した。これらの配列は、クラスタリングアンドアライメントツール（Ｃｌｕｓｔｅｒｉｎｇ ａｎｄ Ａｌｉｇｎｍｅｎｔ Ｔｏｏｌｓ、ＤｏｕｂｌｅＴｗｉｓｔ社、オークランド、ＣＡ）を用い、配列類似性を基にクラスタ化した。各クラスタを含む配列のＧｅｎｂａｎｋ寄託番号は、「Ａｃｃｅｓｓｉｏｎ」欄に列記した。

【０３７７】
【表９Ｂ】表９のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーのゲノム位置及び寄託番号を示す。各々推定されたエキソンについて、本発明者らは、推定に使用したゲノム配列の給源を列記した。各推定されたエキソンのヌクレオチド位置も列記した。

【０３７８】
【表１０】タキソール抵抗性前立腺腫瘍異種移植片においてタキソール感受性前立腺腫瘍異種移植片と比べ示差的に発現された発現配列タグを含む遺伝子を示す。これらの遺伝子は、移植片の逐次継代中にタキソール抵抗性が誘導される間に、アップレギュレーション又はダウンレギュレーションのいずれかであることが示されている。

【０３７９】
【表１１】Ａｆｆｙｍｅｔｒｉｘ／Ｅｏｓ Ｈｕ０１ ＧｅｎｅＣｈｉｐアレイを用いて分析した時、前立腺腫瘍組織において正常前立腺組織と比べアップレギュレーションされた発現配列タグを含む遺伝子を示す。「平均」正常前立腺の「平均」前立腺癌組織に対する比を示した。

【０３８０】
【表１１Ａ】表１１のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーの寄託番号を示す。本発明者らは、各プローブセットについて、そこからオリゴヌクレオチドをデザインした遺伝子クラスタ番号を列記した。遺伝子クラスタは、Ｇｅｎｂａｎｋ ＥＳＴ及びｍＲＮＡ由来の配列を用いて集計した。これらの配列は、クラスタリングアンドアライメントツール（Ｃｌｕｓｔｅｒｉｎｇ ａｎｄ Ａｌｉｇｎｍｅｎｔ Ｔｏｏｌｓ、ＤｏｕｂｌｅＴｗｉｓｔ社、オークランド、ＣＡ）を用い、配列類似性を基にクラスタ化した。各クラスタを含む配列のＧｅｎｂａｎｋ寄託番号は、「Ａｃｃｅｓｓｉｏｎ」欄に列記した。

【０３８１】
【表１２】Ａｆｆｙｍｅｔｒｉｘ／Ｅｏｓ Ｈｕ０１ ＧｅｎｅＣｈｉｐアレイを用いて分析した時、前立腺腫瘍組織において正常前立腺組織と比べダウンレギュレーションされた発現配列タグを含む遺伝子を示す。「平均」正常前立腺の「平均」前立腺癌組織に対する比を示した。

【０３８２】
【表１２Ａ】表１２のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーの寄託番号を示す。本発明者らは、各プローブセットについて、そこからオリゴヌクレオチドをデザインした遺伝子クラスタ番号を列記した。遺伝子クラスタは、Ｇｅｎｂａｎｋ ＥＳＴ及びｍＲＮＡ由来の配列を用いて集計した。これらの配列は、クラスタリングアンドアライメントツール（Ｃｌｕｓｔｅｒｉｎｇ ａｎｄ Ａｌｉｇｎｍｅｎｔ Ｔｏｏｌｓ、ＤｏｕｂｌｅＴｗｉｓｔ社、オークランド、ＣＡ）を用い、配列類似性を基にクラスタ化した。各クラスタを含む配列のＧｅｎｂａｎｋ寄託番号は、「Ａｃｃｅｓｓｉｏｎ」欄に列記した。

【０３８３】
【表１３】Ａｆｆｙｍｅｔｒｉｘ／Ｅｏｓ Ｈｕ０２ ＧｅｎｅＣｈｉｐアレイを用いて分析した時、前立腺腫瘍組織において正常前立腺組織と比べアップレギュレーションされた発現配列タグを含む遺伝子を示す。「平均」正常前立腺の「平均」前立腺癌組織に対する比を示した。

【０３８４】
【表１３Ａ】表１３のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーの寄託番号を示す。本発明者らは、各プローブセットについて、そこからオリゴヌクレオチドをデザインした遺伝子クラスタ番号を列記した。遺伝子クラスタは、Ｇｅｎｂａｎｋ ＥＳＴ及びｍＲＮＡ由来の配列を用いて集計した。これらの配列は、クラスタリングアンドアライメントツール（Ｃｌｕｓｔｅｒｉｎｇ ａｎｄ Ａｌｉｇｎｍｅｎｔ Ｔｏｏｌｓ、ＤｏｕｂｌｅＴｗｉｓｔ社、オークランド、ＣＡ）を用い、配列類似性を基にクラスタ化した。各クラスタを含む配列のＧｅｎｂａｎｋ寄託番号は、「Ａｃｃｅｓｓｉｏｎ」欄に列記した。

【０３８５】
【表１３Ｂ】表１３のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーのゲノム位置及び寄託番号を示す。各々推定されたエキソンについて、本発明者らは、推定に使用したゲノム配列の給源を列記した。各推定されたエキソンのヌクレオチド位置も列記した。

【０３８６】
【表１４】Ａｆｆｙｍｅｔｒｉｘ／Ｅｏｓ Ｈｕ０２ ＧｅｎｅＣｈｉｐアレイを用いて分析した時、前立腺腫瘍組織において正常前立腺組織と比べダウンレギュレーションされた発現配列タグを含む遺伝子を示す。「平均」正常前立腺の「平均」前立腺癌組織に対する比を示した。

【０３８７】
【表１４Ａ】表１４のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーの寄託番号を示す。本発明者らは、各プローブセットについて、そこからオリゴヌクレオチドをデザインした遺伝子クラスタ番号を列記した。遺伝子クラスタは、Ｇｅｎｂａｎｋ ＥＳＴ及びｍＲＮＡ由来の配列を用いて集計した。これらの配列は、クラスタリングアンドアライメントツール（Ｃｌｕｓｔｅｒｉｎｇ ａｎｄ Ａｌｉｇｎｍｅｎｔ Ｔｏｏｌｓ、ＤｏｕｂｌｅＴｗｉｓｔ社、オークランド、ＣＡ）を用い、配列類似性を基にクラスタ化した。各クラスタを含む配列のＧｅｎｂａｎｋ寄託番号は、「Ａｃｃｅｓｓｉｏｎ」欄に列記した。

【０３８８】
【表１４Ｂ】表１４のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーのゲノム位置及び寄託番号を示す。各々推定されたエキソンについて、本発明者らは、推定に使用したゲノム配列給源を列記した。各推定されたエキソンのヌクレオチド位置も列記した。

【０３８９】
【表１５】表１６に示された配列情報を伴う１６９種の遺伝子
表１５は、表１６の配列全てに関する、ｕｎｉｇｅｎｅＩＤ、ｕｎｉｇｅｎｅタイトル、プライムキー、推定された細胞局在、及び典型的寄託番号を示している。表１５の情報は、表１６のＥｏｓＣｏｄｅにリンクしている。

【０３９０】
【表１５Ａ】表１５のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーの寄託番号を示す。本発明者らは、各プローブセットについて、そこからオリゴヌクレオチドをデザインした遺伝子クラスタ番号を列記した。遺伝子クラスタは、Ｇｅｎｂａｎｋ ＥＳＴ及びｍＲＮＡ由来の配列を用いて集計した。これらの配列は、クラスタリングアンドアライメントツール（Ｃｌｕｓｔｅｒｉｎｇ ａｎｄ Ａｌｉｇｎｍｅｎｔ Ｔｏｏｌｓ、ＤｏｕｂｌｅＴｗｉｓｔ社、オークランド、ＣＡ）を用い、配列類似性を基にクラスタ化した。各クラスタを含む配列のＧｅｎｂａｎｋ寄託番号は、「Ａｃｃｅｓｓｉｏｎ」欄に列記した。

【０３９１】
【表１５Ｂ】表１５のｕｎｉｇｅｎｅＩＤを欠いているこれらのプライムキーのゲノム位置及び寄託番号を示す。各々推定されたエキソンについて、本発明者らは、推定に使用したゲノム配列給源を列記した。各推定されたエキソンのヌクレオチド位置も列記した。

【０３９２】
表１１及び配列表

【０３９３】
先に説明した実施例は、いかなる様式においても、本発明の真の範囲を限定するためには利用されず、むしろ例示を目的として示されていることが理解される。各々の刊行物又は特許出願が参照として組入れられることを具体的かつ個別に示されているように、本明細書に引用される全ての刊行物、配列寄託番号、及び特許明細書は、本明細書に参照として組入れられる。[0001]
Cross-reference of related applications
This application claims priority from the following applications: US patent application Ser. No. 09 / 687,576 filed Oct. 13, 2000; U.S. patent filed Mar. 16, 2001. Application 60 / 276,791; US patent application 60 / 288,589 filed May 4, 2001; US patent application 09 / 733,742 filed December 8, 2000; U.S. patent application Ser. No. 09 / 733,288 filed on Dec. 8, 2000; U.S. patent application Ser. No. 09 / 847,046 filed on Apr. 30, 2001; U.S. Patent Application No. 60 / 276,888 filed; U.S. Patent Application No. 60 / 286,214 filed on Apr. 24, 2001; U.S. Patent Application No. 60 filed on Apr. 6, 2001. / 28 , 922; 2001, U.S. Patent Application No. 60 / 263,957, filed January 24, in their entirety are hereby incorporated by reference.
[0002]
TECHNICAL FIELD OF THE INVENTION
The present invention identifies nucleic acid and protein expression profiles associated with prostate cancer and the identification of nucleic acids, products and antibodies thereto; and such expression profiles and compositions in prostate cancer diagnosis, prognosis and treatment Concerning the use of things. The invention further relates to methods of identifying and using substances and / or targets that inhibit prostate cancer.
[0003]
Background of the Invention
Prostate cancer is the most commonly diagnosed medical malignancy in the United States and the second leading cause of cancer death in men, resulting in approximately 40,000 deaths each year (Landis et al., CA Cancer J. Clin., 48: 6-29 (1998); Greenlee et al., CA Cancer J. Clin., 50 (1): 7-13 (2000)), the prevalence of prostate cancer is much worldwide. (Nakata et al., Int. J. Urol., 7 (7): 254-257 (2000); Majeed et al., BJU Int., 85 (9): 1058-). 1062 (2000)). This occurs as a result of pathological malignant transformation of normal prostate cells. With respect to tumor development, the cancer cells undergo a loss of initiation, growth, and contact inhibition, maximally infiltrating surrounding tissues and ultimately metastasize.
[0004]
Death from prostate cancer is the result of prostate tumor metastasis. Therefore, early detection of prostate cancer development is important in reducing mortality from this disease. Measurement of prostate specific antigen (PSA) levels is a very common method for early detection and screening and may have contributed to a slight reduction in prostate cancer mortality in recent years (Nowroozi et al., Cancer Control, 5 (6): 522-531 (1998)). However, many cases have not been diagnosed until the disease has progressed to an advanced stage.
[0005]
Treatments such as surgery (prostatectomy), radiation therapy, and chemotherapy may be curable if the cancer is confined to the prostate. Therefore, early detection of prostate cancer is important for positive diagnosis for treatment. Systemic treatment of metastatic prostate cancer is limited to hormone therapy and chemotherapy. Chemical or surgical castration is the primary treatment for symptomatic metastatic prostate cancer after age 50 years. This testicular androgen depletion therapy usually results in stabilization or regression of the disease (80% of patients) but eventually leads to progression to metastatic prostate cancer (Panvitian et al., Cancer Control, 3 (6): 493-500 (1996)). Metastatic disease is now considered incurable, and the main goals of treatment are life extension and improvement of quality of life (Rago, Cancer Control, 5 (6): 513-521 (1998)).
[0006]
Therefore, a method that can be used for the diagnosis and prognosis of prostate cancer and the effective treatment of prostate cancer, particularly including metastatic prostate cancer, is desired. Accordingly, provided herein are methods that can be used for prostate cancer diagnosis and prognosis. Further provided are methods that can be used to screen candidate bioactive agents for the ability to modulate prostate cancer, eg, the ability to treat prostate cancer. In addition, molecular targets and compositions of therapeutic intervention for prostate cancer and other cancers are provided herein.
[0007]
Summary of the Invention
Thus, the present invention provides the nucleotide sequence of genes that are upregulated and downregulated in prostate cancer cells. Such genes are useful for diagnostic purposes and also as targets for screening therapeutic compounds that modulate prostate cancer, such as hormones or antibodies. Other aspects of the invention will be apparent to those skilled in the art from the following description of the invention.
[0008]
In one aspect, the present invention provides a method of detecting transcripts associated with prostate cancer in cells from a patient, wherein the method comprises a biological sample from a patient with the sequences shown in Tables 1-16. Contacting with a polynucleotide that selectively hybridizes to a sequence that is at least 80% identical.
[0009]
In certain embodiments, the present invention provides a method of detecting the level of transcript associated with prostate cancer in a patient's cells.
[0010]
In certain embodiments, the present invention provides a method of detecting transcripts associated with prostate cancer in a patient's cells, wherein the method comprises subjecting a biological sample from the patient to at least 80 sequences as shown in Tables 1-16. Contacting with a polynucleotide that selectively hybridizes to the% identical sequence.
[0011]
In certain embodiments, the polynucleotide selectively hybridizes to a sequence that is at least 95% identical to the sequence shown in Tables 1-16. In another embodiment, the polynucleotide comprises a sequence shown in Tables 1-16.
[0012]
In certain embodiments, the biological sample is a tissue sample. In another embodiment, the biological sample comprises isolated nucleic acid, such as mRNA.
[0013]
In certain embodiments, the polynucleotide is labeled, eg, with a fluorescent label.
[0014]
In certain embodiments, the polynucleotide is immobilized on a solid surface.
[0015]
In certain embodiments, the patient is undergoing therapeutic medication management to treat prostate cancer. In another embodiment, the patient is suspected of having metastatic prostate cancer.
[0016]
In certain embodiments, the patient is a human.
[0017]
In certain embodiments, the patient is suspected of having taxol-resistant cancer.
[0018]
In some embodiments, the transcript associated with prostate cancer is mRNA.
[0019]
In certain embodiments, the method further comprises amplifying the nucleic acid prior to contacting the biological sample with the polynucleotide.
[0020]
In another aspect, the present invention provides a method for monitoring the efficacy of a therapeutic treatment for prostate cancer, the method comprising: (i) procuring a biological sample from a patient undergoing therapeutic treatment; And (ii) associated with prostate cancer in the biological sample by contacting the biological sample with a polynucleotide that selectively hybridizes to a sequence that is at least 80% identical to a sequence shown in Tables 1-16. Determining the level of transcript and thereby monitoring the efficacy of the therapy. In a further embodiment, the patient has metastatic prostate cancer. In further embodiments, the patient has a drug resistant (eg, taxol resistant) form of prostate cancer.
[0021]
In certain embodiments, the method further comprises: (iii) a level of transcript associated with prostate cancer that is related to the level of transcript associated with prostate cancer in a biological sample from a patient prior to or prior to therapeutic treatment. Including a step of comparing.
[0022]
In addition, a method is provided for evaluating the effects of a drug on a candidate prostate cancer comprising administering the drug to a patient and collecting a cell sample from the patient. The expression profile of the cell is then determined. The method further includes comparing the expression profile with that of a healthy individual. In preferred embodiments, the expression profile comprises the genes of Tables 1-16.
[0023]
In certain aspects, the present invention provides isolated nucleic acid molecules consisting of the polynucleotide sequences shown in Tables 1-16.
[0024]
In certain embodiments, the expression vector or cell comprises an isolated nucleic acid.
[0025]
In certain aspects, the present invention provides isolated polypeptides encoded by nucleic acid molecules having the polynucleotide sequences shown in Tables 1-16.
[0026]
In another aspect, the present invention provides an antibody that specifically binds to an isolated polypeptide encoded by a nucleic acid molecule having a polynucleotide sequence shown in Tables 1-16.
[0027]
In certain embodiments, the antibody is conjugated to an effector component, such as a fluorescent label, a radioisotope, or a cytotoxic chemical.
[0028]
In certain embodiments, the antibody is an antibody fragment. In another embodiment, the antibody is humanized.
[0029]
In one aspect, the present invention provides a method for detecting prostate cancer cells in a biological sample from a patient, the method contacting the biological sample with an antibody as described herein. Including doing.
[0030]
In another aspect, the present invention provides a method for detecting an antibody specific for a patient's prostate cancer, wherein the method comprises analyzing a biological sample from a patient with a nucleic acid comprising a sequence from Tables 1-16. Contacting with the encoded polypeptide.
[0031]
In another aspect, the invention provides a method of identifying a compound that modulates a polypeptide associated with prostate cancer, the method comprising contacting (i) the compound with a polypeptide associated with prostate cancer, wherein Wherein the polypeptide is encoded by a polynucleotide that selectively hybridizes to a sequence that is at least 80% identical to a sequence shown in Tables 1-16; and (ii) a functional action of the compound on the polypeptide. Including the step of determining.
[0032]
In some embodiments, the functional action is a physical action, an enzymatic action or a chemical action.
[0033]
In certain embodiments, the polypeptide is expressed in a eukaryotic host cell or cell membrane. In another embodiment, the polypeptide is recombinant.
[0034]
In certain embodiments, the functional effect is determined by measuring a ligand that binds to the polypeptide.
[0035]
In another aspect, the invention provides a method of inhibiting proliferation of cells associated with prostate cancer to treat prostate cancer in a patient, the method being identified as described herein. Administering to the subject a therapeutically effective amount of a compound.
[0036]
In certain embodiments, the compound is an antibody.
[0037]
In another aspect, the invention provides (i) administering a test compound to a mammal having prostate cancer or a cell sample isolated therefrom; (ii) Tables 1-16 in the treated cell or mammal Comparing the gene expression level of a polynucleotide that selectively hybridizes to a sequence that is at least 80% identical to the sequence shown in to a control cell or mammalian polynucleotide, wherein the polynucleotide expression level A test compound that modulates a drug screening assay is a candidate for the treatment of prostate cancer.
[0038]
In certain embodiments, the control is a mammal with prostate cancer or a cell sample therefrom that has not been treated with the test compound. In another embodiment, the control is a normal cell or mammal.
[0039]
In certain embodiments, the test compound is administered in different amounts or concentrations. In another embodiment, the test compound is administered with varying duration. In another aspect, this comparison can be made after the addition or removal of drug candidates.
[0040]
In certain embodiments, the levels of a plurality of polynucleotides that selectively hybridize to sequences that are at least 80% identical to the sequences shown in Tables 1-16 are individually compared to their respective levels in a control cell sample or mammal. . In a preferred embodiment, the plurality of polynucleotides is 3 to 10 types.
[0041]
In another aspect, the present invention provides a method of treating a mammal having prostate cancer comprising administering a compound identified by the assay methods described herein.
[0042]
In another aspect, the present invention provides a pharmaceutical composition for treating a mammal having prostate cancer, the composition comprising a compound identified by the assay described herein and a physiologically acceptable Contains possible excipients.
[0043]
In one aspect, the present invention provides a method for screening drug candidates by providing cells expressing genes that are up- and down-regulated in prostate cancer. In certain embodiments, the gene is selected from Tables 1-16. The method further includes adding a drug candidate to the cell and determining the effect of the drug candidate on expression of the expression profile gene.
[0044]
In some embodiments, the drug candidate screening method comprises comparing the expression level in the absence of the drug candidate with the expression level in the presence of the drug candidate, where the drug candidate is present. The concentration can be varied, where this comparison can be made after addition or removal of the drug candidate. In a preferred embodiment, the cell expresses at least two expression profile genes. These profile genes may show an increase or a decrease.
[0045]
Further, the action of the candidate prostate cancer drug, comprising the step of administering the drug to a transgenic animal that expresses or overexpresses the prostate cancer modulator protein, or an animal that lacks the prostate cancer modulator protein as a result of, for example, gene knockout. A method for evaluation is also provided.
[0046]
Further provided is a biochip comprising one or more nucleic acid segments of Tables 1-16, wherein the biochip comprises fewer than 1000 nucleic acid probes. Preferably at least two nucleic acid segments are included. More preferably, at least three nucleic acid segments are included.
[0047]
Further provided are methods of diagnosing diseases associated with prostate cancer. The method determines the gene expression of Tables 1-16 in a first tissue type of a first individual and distributes this distribution to a second from a first individual or a second unaffected individual. Comparing with normal tissue type gene expression. Differences in expression indicate that the first individual has a disease associated with prostate cancer.
[0048]
In further embodiments, the biochip further comprises a polynucleotide sequence of a gene that is not up-regulated and down-regulated in prostate cancer.
[0049]
In some embodiments, the method comprises a bioactive agent capable of interfering with binding to a protein that modulates prostate cancer (prostate cancer modulator protein) or a fragment thereof and an antibody that binds to the prostate cancer modulator protein or a fragment thereof. It is a screening method. In a preferred embodiment, the method comprises combining a prostate cancer modulator protein or fragment thereof with a candidate bioactive agent and antibody that binds to the prostate cancer modulator protein or fragment thereof. The method further includes determining binding of the prostate cancer modulator protein or fragment thereof to the antibody. If there is a change in binding, the substance is identified as an interfering substance. The interfering substance can be an agonist or an antagonist. Preferably the substance inhibits prostate cancer.
[0050]
Further provided herein are methods for eliciting an immune response in an individual. In certain embodiments, the methods provided herein comprise administering to an individual a composition containing prostate cancer modulating protein, or a fragment thereof. In another embodiment, the protein is encoded by a nucleic acid selected from Tables 1-16.
[0051]
Further provided herein are compositions that are capable of eliciting an immune response in an individual. In certain embodiments, the compositions provided herein contain a prostate cancer modulating protein or fragment thereof, preferably encoded by the nucleic acids of Tables 1-16, and a pharmaceutically acceptable carrier. In another embodiment, the composition comprises a nucleic acid comprising a sequence encoding a prostate cancer modulating protein, preferably selected from the nucleic acids of Tables 1-16, and a pharmaceutically acceptable carrier.
[0052]
Also provided is a method of neutralizing the action of prostate cancer proteins or fragments thereof, comprising contacting a protein specific substance with the protein in an amount sufficient to effect neutralization. In another embodiment, the protein is encoded by a nucleic acid selected from those in Tables 1-16.
[0053]
In another aspect of the invention, a method of treating an individual for prostate cancer is provided. In certain embodiments, the method comprises administering to the individual an inhibitor of prostate cancer modulating protein. In another embodiment, the method includes administering to a patient with prostate cancer an antibody against prostate cancer modulating protein conjugated to a therapeutic moiety. Such therapeutic moiety can be a cytotoxic substance or a radioisotope.
[0054]
Detailed Description of the Invention
In accordance with the objects outlined above, the present invention provides a novel method for the diagnosis and prognostic assessment of prostate cancer (PC), including metastatic prostate cancer, as well as a method of screening for compositions that modulate prostate cancer. . Also provided are methods of treating prostate cancer.
[0055]
The present invention relates to the identification of PAA2 as a gene that is highly overexpressed in prostate cancer patient tissues in addition to other nucleic acid and peptide sequences. The PAA2 sequence is the same as the zinc transporter ZNT4. From the results described herein, it is clear that PAA2 / ZNT4 is highly expressed in prostate cancer cells. The prostate is unique in that it has the highest zinc accumulation capacity in the organs of the body. Zinc uptake is regulated by prolactin and testosterone, which induces expression of members of the ZIP family of zinc transporters (Costello et al., J. Biol. Chem., 274: 17499-17504 (1999)). Zinc accumulation in the prostate functions to inhibit citrate oxidation, which results in a decrease in cellular ATP production (Costello and Franklin, Prostate, 35: 285-296 (1998)). Cancer cells are more sensitive to reduced ATP production and develop into interfering with zinc accumulation. Without wishing to be theoretically supported, up-regulation of ZNT4 in prostate cancer cells results in protection of cells from high zinc levels by virtue of their ability to pump accumulated zinc from the cells.
[0056]
The invention also relates to a nucleic acid sequence encoding PBH1. PBH1 is a putative calcium channel highly expressed in the brain, human TRPC7 (transient receptor potential-related channels, NP_003298) (Nagamine et al., Genomics, 54: 124- 131 (1998)). Trp is localized in the affected area of melastatin (Duncan et al., Cancer Res., 58: 1515-1520 (1998)), a down-regulated gene in metastatic melanoma, and Bekwis-Bidemann syndrome / Wilmus tumor It is related to MTR1, which is an active gene (Prawitt et al., Hum. Mol. Genet., 9: 203-216 (2000)). Without wishing for theoretical support, PBH1 is thought to function as a calcium channel.
[0057]
PBH1 as a calcium channel is an ideal target for small molecule therapy or therapeutic antibodies that disrupt channel function. CD2O (Maloney et al., Blood, 90: 2188-2195 (1997); Leget and Czuczman, Curr. Opin. Oncol., 10: 548-551 (1998)) is a target of Rituximab in non-Hodgkin lymphoma It is an expressed plasma membrane calcium channel (Tedder and Engel, Immunol. Today, 15: 450-454 (1994)). Similarly, small molecules or antibodies that inhibit or alter the calcium signal will be mediated by PBH1, leading to prostate cancer cell death.
[0058]
PEH1 and other genes of the present invention are also useful as targets for cytotoxic T lymphocytes. Genes that are tumor specific or expressed in immune privileged organs are currently used as potential vaccine targets (Van den Eynde and Boon, Int. J. Clin. Lab. Res., 27: 81-86 (1997)). The expression pattern of PBH1 indicates that this is an ideal target for cytotoxic T-lymphocytes. Accordingly, therapies that utilize PBH1-specific cytotoxic T lymphocytes to induce prostate cancer cell death are also provided in the present invention. See, for example, US Pat. No. 6,051,227 and International Publication No. WO 00/32231, the disclosures of which are incorporated herein by reference.
[0059]
The invention also relates to the identification of PAA3 as a gene that is important in the modulation of prostate cancer and / or breast cancer.
[0060]
Tables 1-16 provide Unigene cluster identification numbers, typical accession numbers, or genomic nucleotide position numbers for genes that show increased or decreased expression in prostate cancer samples.
[0061]
Definition
The term “prostate cancer protein” or “prostate cancer polynucleotide” or “transcript associated with prostate cancer” refers to (1) the nucleotide sequence of the unigene cluster of Tables 1-16 or the nucleotide sequence associated therewith, preferably For regions of at least about 25, 50, 100, 200, 500, 1000 or more nucleotides, greater than about 60% nucleotide sequence identity, 65%, 70%, 75%, 80%, 85%, 90% Preferably having a nucleotide sequence having nucleotide sequence identity of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater; (2) Table A sequence encoded by the nucleotide sequence of 1 to 16 unigene clusters or a nucleotide sequence related thereto. Binding to an antibody raised against an immunogen comprising a nonacid sequence, such as a polyclonal antibody, and conservatively modifying their variants; (3) nucleic acid sequences of Tables 1-16, or their complements and conservatives Specifically hybridize under stringent hybridization conditions; or (4) encoded by the nucleotide sequence of the unigene cluster of Tables 1-16 or a nucleotide sequence related thereto. More than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85 for amino acids, preferably for at least about 25, 50, 100, 200, 500, 1000 or more amino acid regions %, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% Such as has an amino acid sequence having more amino sequence identity than that polymorphic variants of nucleic acids and polypeptides, allelic variants, refers to interspecies homologs. The polynucleotide or polypeptide sequence typically includes, but is not limited to, primates such as humans; rodents such as rats, mice, hamsters; cows, pigs, horses, sheep, or other mammals. It comes from a mammal that is not a thing. “Prostate cancer polypeptides” and “prostate cancer polynucleotides” include both natural or recombinant forms.
[0062]
A “full length” prostate cancer protein or nucleic acid comprises a prostate cancer polypeptide or polynucleotide sequence comprising all of the elements normally contained in one or more natural wild-type prostate cancer polynucleotide or polypeptide sequences; Or a variant thereof. For example, a full length prostate cancer nucleic acid will typically include all of the exons that encode the full length native protein. “Full length” can be before or after various stages of post-translational processing or splicing, including alternative splicing.
[0063]
As used herein, a “biological sample” is a sample of biological tissue or fluid containing nucleic acids or polypeptides, such as prostate cancer proteins, polynucleotides or transcripts. Such samples include, but are not limited to, tissues isolated from primates, such as humans, or rodents, such as mice and rats. Biological samples also include tissue sections such as biopsy samples and autopsy samples, frozen sections taken for histological purposes, blood, plasma, serum, sputum, feces, tears, mucous membranes, hair, skin, etc. . Biological samples further include explants derived from patient tissue and primary and / or transformed cell cultures. The biological sample is typically from a eukaryote, most preferably a mammal, eg a primate such as a chimpanzee or a human; a cow; a dog; a cat; a rodent such as a guinea pig, rat, mouse; Or from birds; reptiles; or fish.
[0064]
“Providing a biological sample” means obtaining a biological sample for use in the methods described in the present invention. Most often, this is done by taking a cell sample from the animal, but pre-isolated cells (eg, isolated from another human, another time point, and / or for another purpose). Or by carrying out the method of the invention in vivo. Accumulated tissue with a history of treatment or outcome will be particularly useful.
[0065]
In reference to two or more nucleic acid or polypeptide sequences, the term “identical” or% “identity” uses the BLAST or BLAST 2.0 sequence comparison algorithm with the following default parameters: Specific percentages of amino acid residues or nucleotides that are the same or the same (ie, for a comparison window or for a specified region) When compared and juxtaposed, for a particular region, about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity) Means a partial sequence (see, eg, NCBI website, http://www.ncbi.nlm.nih.gov/BLAST/, etc.). Such sequences are then referred to as “substantially the same”. This identification is also referred to as, or can be applied to, the complement of the test sequence. This definition further includes those having substitutions in addition to sequences having deletions and / or additions, as well as natural, eg polymorphic or allelic variants, and artificial variants. As described below, preferred algorithms can account for gaps and the like. Preferably, identity exists for a region that is at least about 25 amino acids or nucleotides in length, or more preferably for a region that is 50-100 amino acids or nucleotides in length.
[0066]
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence relative to the reference sequence, based on the program parameters.
[0067]
As used herein, a “comparison window” is typically a series selected from the group consisting of 20 to 600, usually about 50 to about 200, more commonly about 100 to about 150. Reference to a single segment of the number of positions is included, in which, after optimal alignment of the two sequences, the sequence is compared to a reference sequence of the same number of consecutive positions. Sequence alignment methods for comparison are well known in the art. Optimal alignment of the sequences for comparison is, for example, Smith and Waterman's local homology algorithm (Adv. Appl. Math., 2: 482 (1981)), Needleman and Wunsch. ) Homology alignment algorithm (J. Mol. Biol., 48: 443 (1970)), Pearson and Lipman similarity search (Proc. Natl. Acad. Sci. USA, 85: 2444 ( 1988)), computer implementation of these algorithms (GAP, BESTFLT, FASTA, and TFASTA, Ge in Wisconsin Genetics Software Package) etics Computer Group, 575 Science Dr., Madison, WI), or by manual juxtaposition and visual inspection (eg, “Current Protocols in Molecular Biology” (edited by Ausubel et al., 1995). Year, addendum)).
[0068]
Preferred examples of algorithms that are suitable for determining% sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (Nuc. Acids Res., 25: 3389-3402). (1977)) and Altschul et al. (J. Mol. Biol., 215: 403410 (1990)). BLAST and BLAST 2.0 are used with the parameters noted herein to determine the percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyzes is publicly available from the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/). This algorithm initially uses a query sequence that either matches or satisfies several positive threshold scores T when juxtaposed with words of the same length in the database sequence. Identifying high scoring segment pairs (HSPs) by identifying short words of length W. T is referred to as the neighborhood word score threshold (Altschul et al., Supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. These word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. The cumulative score is calculated, for example for nucleotide sequences, using the parameters M (reward score of matching residue pair; always> 0) and N (penalty score of mismatched residue; always <0). The For the amino acid sequence, a cumulative score was calculated using a score matrix. The expansion of word hits in each direction was stopped when: the cumulative alignment score fell X amounts from its maximum achieved value; for one or more negative-scoring residue alignments When the cumulative score is zero or less; or when either end of the sequence is reached. The BLAST algorithm parameters W, T and X determine the juxtaposition sensitivity and speed. The BLASTN program (for nucleotide sequences) used word length (W) 11, expectation (E) 10, M = 5, N = -4 and comparison of both strands as default. For amino acid sequences, the BLASTP program uses, as a default, a word length of 3, and an expected value (E) of 10, and a BLOSUM62 score matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89: 10915 (1989). Reference) Alignment (B) 50, expected value (E) 10, M = 5, N = -4, and comparison of both strands was used.
[0069]
The BLAST algorithm also performed statistical analysis on the similarity between the two sequences (see, eg, Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which gives an indication of the probability that a match will occur by chance between two nucleotide or amino acid sequences. Provided. For example, a nucleic acid is similar to a reference sequence if the minimum sum probability in the comparison of the test nucleic acid and the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001. Is considered to be. The logarithmic value is larger than the negative number, for example, 5, 10, 20, 30, 40, 40, 70, 90, 110, 150, 170, and the like.
[0070]
Identification of two nucleic acid sequences or polypeptides that are substantially the same resulted in a polypeptide encoded by the first nucleic acid relative to the polypeptide encoded by the second nucleic acid, as described below. It is immunologically cross-reactive with antibodies. Thus, for example, if these two peptides differ only by conservative substitutions, the polypeptide is typically substantially the same as the second polypeptide. Another indication that two nucleic acid sequences are substantially the same is that two molecules or their complements hybridize to each other under stringent conditions, as explained below. Yet another indication that two nucleic acid sequences are substantially the same is that the same primers can be used to amplify these sequences.
[0071]
A “host cell” is a natural cell or a transformed cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells can be cultured cells, explants, cells in vivo, and the like. The host cell may be a prokaryotic cell such as E. coli or a eukaryotic cell such as a yeast, insect, amphibian or mammalian cell, examples of which include CHO, HeLa, etc. (eg, American Type Culture Collection catalog or website (see www.atcc.org).
[0072]
The terms "isolated", "purified" or "biologically pure" refer to materials that are substantially or essentially free from components that are normally absent as found in their native state. means. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. Proteins or nucleic acids that are the major species present in the preparation are substantially purified. In particular, an isolated nucleic acid has been separated from some open reading frame that naturally flanks a gene and encodes a protein other than the protein encoded by the gene. In some embodiments, the term “purified” means that the nucleic acid or protein appears in essentially one band in the electrophoresis gel. Preferably, this means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. In another embodiment, “purify” or “purification” means removing at least one contaminant from the composition to be purified. In this sense, purification does not require the purified compound to be homogeneous, for example 100% pure.
[0073]
The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to mean a polymer of amino acid residues. These terms include residues modified with natural amino acid polymers in addition to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding natural amino acids, and non- Applies to those containing natural amino acid polymers.
[0074]
The term “amino acid” refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Natural amino acids include those encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds having the same basic chemical structure as natural amino acids, for example, hydrogen, carboxyl group, amino group, and α-carbon bonded to R group, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, etc. means. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics means a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a natural amino acid.
[0075]
In the present specification, amino acids can be represented by either their commonly known three-letter code or by the one-letter code recommended by the Biochemical Nomenclature Commission of IUPAC-IUB. . Similarly, nucleotides can be represented by the normally accepted single letter code.
[0076]
“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. For a particular nucleic acid sequence, conservatively modified variants are essentially the same if the nucleic acid encodes the same or essentially the same amino acid sequence, or if the nucleic acid does not encode an amino acid sequence. Or refer to related sequences, such as natural contig sequences. Because of the degeneracy of the genetic code, most proteins are encoded by a large number of functionally identical nucleic acids. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where an alanine is specified by a codon, the codon can be changed to another corresponding codon as described without changing the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Any nucleic acid sequence encoding a polypeptide herein describes a silent mutation of the nucleic acid. One skilled in the art, in some circumstances, modifies each codon of the nucleic acid (except AUG, which is usually only a codon for methionine, and TGG, which is usually only a codon for tryptophan) to obtain a functionally identical molecule. You will admit that you can. Thus, silent mutations of nucleic acids encoding polypeptides are implicit in the sequence described for the expression product, but often not the actual probe sequence.
[0077]
As far as the amino acid sequence is concerned, those skilled in the art will recognize individual substitutions, deletions in nucleic acid, peptide, polypeptide or protein sequences that alter, add or delete one amino acid or a small percentage of amino acids in the encoded sequence. Or addition will recognize that this change is a “conservatively modified variant” resulting in a substitution of a chemically similar amino acid for an amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are added to, but do not exclude, polymorphic variants, interspecies homologues, and alleles of the present invention and typically include Substitution: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine ( K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine (C), methionine (M) (see, eg, Creighton's “Proteins” (1984)).
[0078]
Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For general considerations regarding this organization, see, for example, Alberts et al., “Molecular Biology of the Cell (3rd Edition, 1994)” and “Cantor and Schimmel” See Biophysical Chemistry, Part I: Conformation of biological macromolecules (1980) “Primary structure” means the amino acid sequence of a particular peptide. “Means a three-dimensional structure arranged locally within a polypeptide. These structures are usually known as domains. Domains often form a compact unit of the polypeptide. Part of a polypeptide, typically 25 to about 5 A typical domain is made up of less organized compartments, such as β-sheet and α-helix stretches, “tertiary structure” means the complete three-dimensional structure of a polypeptide monomer “Quaternary structure” means a three-dimensional structure usually composed of non-covalent associations of independent three-dimensional units, and the term anisotropic is also known as an energy term.
[0079]
As used herein, “nucleic acid” or “oligonucleotide” or “polynucleotide” or grammatical equivalent means at least two nucleotides covalently linked together. Oligonucleotides are typically about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides in length, up to about 100 nucleotides in length . Nucleic acids and polynucleotides are polymers of any length, including longer lengths such as 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. The nucleic acids of the invention generally comprise a phosphodiester linkage, but in some cases, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O-methyl phosphoramidate linkages (Eckstein, “Oligonucleotides and analogs: practice Nucleic acid analogs that may have alternative backbones, including peptide nucleic acid backbones, linkages, and the like; and the like (Oligonucleotide and Analogues: A Practical Approach), Oxford University Press) Other nucleic acid analogs include nucleic acid analogs with a positive backbone; a nonionic backbone, and a non-ribose backbone, which are described in US Pat. Nos. 5,235,033 and 5,034,506. As well as nucleic acid analogues described in ASC Symposium Series 580 “Carbohydrate Modifications in Antisense Research” chapters 6 and 7, edited by Sanghui and Cook including. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acid. Modification of the ribose-phosphate backbone can be done for a variety of reasons, for example, to increase the stability and half-life of such molecules in a physiological environment, or as a biochip probe. Natural nucleic acid and analog mixtures can be made; alternatively, various nucleic acid analog mixtures and natural nucleic acid and analog mixtures can be made.
[0080]
Various references regarding such nucleic acid analogs have been identified, including, for example, phosphoramidate linkages (Beaucage et al., Tetrahedron, 49 (10): 1925 (1993) and references therein; Letsinger, J. Org. Chem., 35: 3800 (1970); Sprinzl et al., Eur. J. Biochem., 81: 579 (1977); Letsinger et al., Nucl. Acids Res., 14: 3487 (1986); Chem. Lett., 805 (1984), Letsinger et al., J. Am. Chem. Soc., 110: 4470 (1988); and Pauwels et al., Chemica Scripta, 26: 141 (1986)), phospho. Thioate linkage (Mag et al., Nucleic Acids Res., 19: 1437 (1991); and US Pat. No. 5,644,048), phosphorodithioate linkage (Briu et al., J. Am. Chem. Soc., 111 : 2321 (1989)), O-methyl phosphoramidite linkage (see Eckstein, “Oligonucleotides and Analogues: A Practical Method”, Oxford University Press and Peptide Nucleic Acid) Binding (Egholm, J. Am. Chem. Soc., 114: 1895 (1992); Meier et al., Chem. Int. Ed. Engl., 31: 1008 (199) Including 207 (1996), which is incorporated by reference in full herein): Carlsson et al, Nature, 380; 566 (1993):;) Nielsen, Nature, 365. Other nucleic acid analogs include a positive backbone (Denpcy et al., Proc. Natl. Acad. Sci. USA, 92: 6097 (1995)); a nonionic backbone (US Pat. No. 5,386,023, 5, 637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. England, 30: 423 (1991); J. Am. Chem. Soc., 110: 4470 (1988); Letsinger et al., Nucleoside & Nucleotide, 13: 1597 (1994); ASC Symposium Series 580, “Carbohydrate Modifiers in Antisense Searches”. 2) and Chapter 3 edited by YS Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett., 4: 395 (1994); Jeffs et al, c. : 17 (1994); Tetrahedron Lett., 37: 743 (1996)), and non-ribose backbones, US Pat. Nos. 5,235,033 and 5,034,506, and ASC Symposium Series 580, “ Sugar Modifications in Antisense Search (Carbohydrate Modifications in Antisense Research) "Chapters 6 and 7, S. Sanghui and Y. Sanghui Including those described in P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included in one definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev., 169-176 (1995)). Several nucleic acid analogs are described in Rawls, C & E News, June 2, 1997, page 35. All of these references are expressly incorporated herein by reference.
[0081]
Particularly preferred are peptide nucleic acids (PNA) comprising peptide nucleic acid analogs. In contrast to the highly charged phosphodiester backbones of natural nucleic acids, these backbones are substantially nonionic under neutral conditions. This produces two advantages. First, the PNA backbone exhibits improved hybridization kinetics. PNA has a melting point (T_m) With greater variation. DNA and RNA typically have T_m2 to 4 ° C. decrease. This decrease due to the nonionic PNA skeleton is close to 7-9 ° C. Similarly, due to their nonionic nature, the hybridization of bases attached to these backbones is relatively insensitive to salt concentrations. In addition, PNA is not degraded by cellular enzymes and as a result can be more stable.
[0082]
Nucleic acids can be single-stranded or double-stranded, or can include portions of both single-stranded or double-stranded sequences. As will be appreciated by those skilled in the art, the description of a single strand also defines the sequence of the complementary strand; therefore, the sequences described herein also provide the complement of that sequence. This nucleic acid is a hybrid in which both genomic and cDNA DNA, RNA or nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides, and uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine Or a combination of bases including isoguanine and the like. “Transcript” typically means natural RNA, such as pre-mRNA, hnRNA, or mRNA. As used herein, the term “nucleoside” includes nucleotides and modified nucleosides such as nucleoside analogs and nucleotide analogs, as well as amino-modified nucleosides. In addition, “nucleoside” includes non-natural analog structures. Thus, for example, individual units of peptide nucleic acids, each containing a base, are referred to herein as nucleosides.
[0083]
A “label” or “detectable moiety” is a composition detectable by microscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, useful labels have been made detectably by the incorporation of a fluorescent dye, a high electron density reagent, an enzyme (eg, commonly used in an ELISA), biotin, digoxigenin, or a hapten and, for example, a radioactive label into a peptide or Includes proteins or other entities such as those used to detect antibodies that specifically react with this peptide. The radioisotope can be, for example, 3H, 14C, 32P, 35S, or 125I. In some cases, particularly with antibodies against the proteins of the invention, these radioisotopes can be used as toxic moieties, as described below. These labels can be incorporated into prostate cancer nucleic acids, proteins and antibodies at any position. Any method known in the art for conjugating antibodies to labels can be used, including Hunter et al., Nature, 144: 945 (1962); David et al., Biochemistry, 13: 1014 (1974); Pain et al., J. MoI. Immunol. Meth. 40: 219 (1981); and Nygren, J. et al. Histochem. and Cytochem. 30: 4O7 (1982). The lifetime of a radiolabeled peptide or radiolabeled antibody composition can be extended by the addition of substances that stabilize the radiolabeled peptide or antibody and interfere with its degradation. Any substance or combination of substances that stabilize radiolabeled peptides or antibodies can be used, including those disclosed in US Pat. No. 5,961,955.
[0084]
“Effector” or “effector moiety” or “effector component” is an antibody, covalently through a linker or chemical bond, or non-covalently through an ionic, van der Waals, electrostatic or hydrogen bond Molecule bound to (or linked to or complexed with). An “effector” is, for example, a radioactive compound, a fluorescent compound, an enzyme or substrate, a tag such as an epitope tag, a detection moiety containing a toxin; an activating moiety such as a chemotherapeutic agent; a lipase; an antibiotic; (Hard) "can be a variety of molecules including radioisotopes that emit beta rays.
[0085]
A “labeled nucleic acid probe or oligonucleotide” is a covalent or ionic bond, van der Waals via a linker or chemical bond, such that the presence of the probe is detected by detection of the presence of the label bound to the probe. It is bound to the label non-covalently through a bond, electrostatic bond or hydrogen bond. Alternatively, methods using high affinity interactions can achieve the same result, where one of the binding partner pairs binds to the other, eg, biotin, streptavidin.
[0086]
As used herein, a “nucleic acid probe or oligonucleotide” is a target of a complementary sequence through one or more chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. Defined as nucleic acid capable of binding to nucleic acid. As used herein, a probe can include a natural base (ie, A, G, C, or T) or a modified base (7-deazaguanosine, inosine, etc.). In addition, the bases in the probe are linked by bonds other than phosphodiester bonds, as long as they do not functionally interfere with hybridization. Thus, for example, a probe can be a peptide nucleic acid in which constitutive bases are linked by peptide bonds other than phosphodiester bonds. One skilled in the art will appreciate that these probes can bind to a target sequence that lacks complete complementarity with the probe sequence, depending on the stringency of the hybridization conditions. These probes are preferably labeled directly with isotopes, chromophores, luminophores, chromogens, or indirectly with, for example, biotin to which the streptavidin complex later binds. By assaying for the presence or absence of the probe, the presence or absence of the selected sequence or subsequence can be detected. Diagnosis or prognosis can be based at the genomic level or at the RNA or protein expression level.
[0087]
The term “recombinant” when used with respect to a cell or nucleic acid, protein or vector, the cell, nucleic acid, protein or vector is modified by introduction of a heterologous nucleic acid or protein or modification of a native nucleic acid or protein. Or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in native (non-recombinant) cells, or are otherwise aberrantly expressed, underexpressed or not expressed at all. Unexpressed native gene is expressed. As used herein, the term “recombinant nucleic acid” refers to a nucleic acid originally formed in vitro in a form not normally found in nature, generally by manipulation of the nucleic acid, eg, the use of polymerases and endonucleases. In this way, an operable linkage of different sequences is realized. Thus, both linearly isolated nucleic acids or expression vectors formed in vitro by ligation of DNA molecules that are not normally ligated are both considered recombinant for the purposes of the present invention. Once a recombinant nucleic acid has been created and reintroduced into a host cell or organism, it replicates non-recombinantly, ie, using the in vivo cellular machinery of the host cell rather than in vitro manipulation; It will be understood that such nucleic acids, once produced recombinantly and then non-recombinantly replicated, are still considered recombinant for the purposes of the present invention. Similarly, a “recombinant protein” is a protein produced using recombinant techniques, ie by expression of the aforementioned recombinant nucleic acids.
[0088]
The term “heterologous” when used with reference to a portion of a nucleic acid indicates that the nucleic acid contains two or more subsequences that are not normally found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, such as promoters from one source and coding regions from another source, eg, unrelated genes arranged to create new functional nucleic acids. It has two or more sequences derived from it. Similarly, a heterologous protein often refers to two or more subsequences that are not found in the same relationship to each other in nature (eg, a fusion protein).
[0089]
A “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the transcription start site, such as a TATA element in the case of a polymerase type II promoter. A promoter can also optionally include a distal enhancer or repressor element, which is located at a base pair thousands of distances from the transcription start site. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operable linkage” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter or an array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence comprises Directs transcription of the nucleic acid corresponding to the second sequence.
[0090]
An “expression vector” is a recombinantly or synthetically created nucleic acid construct with a series of specialized nucleic acid elements that allow transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus or nucleic acid fragment. Typically, an expression vector comprises a nucleic acid to be transcribed operably linked to a promoter.
[0091]
The phrase “selectively (or specifically) hybridizes” refers to nucleotide sequences in a heterogeneous population of nucleic acids and other biologicals (eg, total cells or a library of DNA or RNA). It means that a molecule binds, duplexes or hybridizes to a specific nucleotide sequence that is determinative of presence. Similarly, the phrase “specific (or selective) binding” or “specific (or selective) immune response” to an antibody refers to a heterogeneous population of proteins and other biological things when referring to proteins or peptides. Means a binding reaction that is a determinant of the presence of a protein. Thus, under designated immunoassay conditions or nucleic acid hybridization conditions, a specified antibody or nucleic acid probe will bind to a particular protein nucleotide sequence at least twice background, more typically 10 to 10 background. Combine at 100 times.
[0092]
Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, selecting a polyclonal antibody raised against a specific protein, polymorphic variant, allele, ortholog, and conservatively modified variant, or splicing variant, or part thereof, and a desired prostate cancer protein Only polyclonal antibodies that are specifically immunoreactive but not other proteins can be obtained. This selection can be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats can be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid phase ELISA immunoassay methods are routinely used to select antibodies that are specifically immunoreactive with proteins (immunoassay formats that can be used to determine specific immunoreactivity and For an explanation of the conditions, see, for example, Harlow and Lane, “Antibodies: Experiments, A Laboratory Manual” (1988)).
[0093]
The phrase “stringent hybridization conditions” refers to conditions in which a probe typically hybridizes to its target subsequence in a complex mixture of nucleic acids but not to other sequences. Stringent conditions are determined by the sequence and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Extensive guidelines for nucleic acid hybridization can be found in Tijsssen, “Biochemistry and Molecular Biology Technology—Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes,” See "Overview of principals of hybridization and the sexuality of assays" (1993). In general, stringent conditions are such that the melting point of a specific sequence (T_m) About 5-10 ° C. This T_mIs the temperature (temperature at the specified ionic strength, pH and nucleic acid concentration) at which 50% of the probe complementary to the target hybridizes to the target sequence when equilibrated (T_mThe target sequence is present in excess and 50% of the probe is occupied at the time of equilibration). Stringent conditions are when the salt concentration is less than about 1.0M sodium ion, typically about 0.01-1.0M sodium ion concentration (or other salt) at pH 7.0-8.3. And as low as about 30 ° C. for short probes (eg, 10-50 nucleotides) and at least about 60 ° C. for long probes (eg, longer than 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least twice background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions are: 50% formamide, 5 × SSC, and 1% SDS, 42 ° C. incubation, or 5 × SSC, 1% SDS, 65 ° C. incubation, 0.2 × SSC. Wash, 0.1% SDS at 65 ° C. For PCR, a temperature of about 36 ° C. is typically low stringency amplification, but the annealing temperature can vary from about 32 ° C. to 48 ° C. depending on the primer length. For high stringency PCR amplification, a temperature of about 62 ° C is typical, but high stringency annealing temperatures can range from about 50 ° C to about 65 ° C, depending on primer length and specificity. Typically, cycling conditions for both high and low stringency amplification are: 90 ° C. to 95 ° C. for 30 seconds to 2 minutes of denaturing phase, 30 seconds to 2 minutes of annealing phase, and about 72 ° C. for 1 to 2 Contains an extended phase of minutes. Protocols and guidelines for low and high stringency amplification reactions are described, for example, in Innis et al., “PCR Protocols: Methods and Applications (PCR Protocols, A Guide to Methods and Applications), (1990), Academic Press, NY ).
[0094]
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially the same if the polypeptides that they encode are substantially the same. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy provided by the genetic code. In such cases, the nucleic acid typically hybridizes under moderately stringent hybridization conditions. For example, “moderately stringent hybridization conditions” include hybridization at 37 ° C. in 40% formamide, 1M NaCl, 1% SDS buffer, and washing at 45 ° C. in 1 × SSC. Positive hybridization is at least twice background. Those skilled in the art will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidance regarding the determination of hybridization parameters is provided in a number of references, for example as described in Ausubel et al., "Current Protocols in Molecular Biology".
[0095]
In the context of assays relating to the testing of compounds that modulate the activity of prostate cancer proteins, the phrase “functional effects” refers to, for example, functional, physical or chemical effects such as the ability to reduce prostate cancer. Determining parameters under indirect or direct influence of prostate cancer protein or nucleic acid. This includes ligand binding activity; cell growth on soft agar; anchorage dependence; growth contact inhibition and density limits; cell proliferation; cell malignant transformation; growth factor or serum dependence; tumor-specific marker levels; Matrigel In vivo tumor growth and metastasis; mRNA and protein expression in cells undergoing metastasis, and other characteristics of prostate cancer cells. “Functional effects” include in vitro, in vivo, and ex vivo activities.
[0096]
“Determining a functional effect” refers to assaying a compound that increases or decreases a parameter under the indirect or direct influence of a prostate cancer protein sequence, such as a functional, enzymatic, physical or chemical effect. It means to do. Such functional action may be achieved by any means known to those skilled in the art, such as protein spectroscopic features (eg fluorescence, absorbance, refractive index), hydrodynamic features (eg shape), chromatography or Can be determined by changes in solubility properties, measurement of inducible markers or transcriptional activation of prostate cancer proteins; measurement of binding activity or binding assays such as binding to antibodies or other ligands, and measurement of cell proliferation . Determination of the functional effect of a compound on prostate cancer can also be performed using a prostate cancer assay, such as an in vitro assay known to those skilled in the art, for example, cell growth on soft agar; anchorage dependence; Contact inhibition and density limits; cell proliferation; malignant transformation of cells; growth factor or serum dependence; tumor-specific marker levels; invasiveness to Matrigel; tumor growth and metastasis in vivo; mRNA and protein expression in cells undergoing metastasis And other features of prostate cancer cells. This functional effect can be assessed by a number of means known to those skilled in the art, such as microscopy for quantitative or qualitative measurement of changes in morphological characteristics, RNA of sequences associated with prostate cancer, or Downstream or reporter gene expression (CAT, luciferase, β, by measuring changes in protein level, measuring RNA stability, eg chemiluminescence, fluorescence, colorimetric reaction, antibody binding, inducible markers, and ligand binding assays -Gal, GFP, etc.).
[0097]
“Inhibitors,” “activators,” and “modulators” of prostate cancer polynucleotide and polypeptide sequences are molecules or compounds identified using in vitro and in vivo assays of prostate cancer polynucleotide and polypeptide sequences. Used for activation, inhibition or modulation. Inhibitors, for example, compounds that bind to prostate cancer protein, partially or wholly block activity, reduce, interfere with, delay activation, deactivate, desensitize, or down regulate activity or expression, such as antagonists It is. Antisense nucleic acids appear to inhibit protein expression and subsequent function. “Activators” are compounds that increase, open, activate, promote, enhance activity, sensitize, actuate or upregulate prostate cancer protein activity. Inhibitors, activators or modulators also include genetically modified forms of prostate cancer proteins, such as those with altered activity, as well as natural and synthetic ligands, antagonists, agonists, antibodies, small chemical molecules, and the like. Such inhibitor and activator assays include, for example, expressing prostate cancer proteins in vitro, intracellularly or in the plasma membrane, applying putative modulator compounds, and then functional effects on activity as described above. Including deciding. Prostate cancer cells are incubated with a test compound and one or more prostate cancer proteins such as 1, 2, 3, 4, 5, 10 such as the prostate cancer proteins encoded by the sequences listed in Tables 1-16. , 15, 20, 25, 30, 40, 50 or more prostate cancer protein activators and inhibitors can be identified by determining increased or decreased expression.
[0098]
A sample or assay comprising a prostate cancer protein comprising treatment with a potential activator, inhibitor, or modulator is used to test a degree of inhibition with a control sample that does not contain an inhibitor, activator, or modulator. To be compared. Control samples (untreated with inhibitor) are assigned a relative protein activity value of 100%. Inhibition of the polypeptide is achieved when the activity value relative to the control is about 80%, preferably 50%, more preferably 25-0%. Activation of the prostate cancer polypeptide has an activity value of 110%, more preferably 150%, more preferably 200-500% (ie 2-5 times higher than the control) relative to the control (untreated with activator). More preferably, it is achieved when the content is 1000 to 3000% higher.
[0099]
The phrase “change in cell growth” refers to growth foci formation), anchorage independence, semi-solid or soft agar growth, growth contact inhibition and density limit changes, growth factor or serum requirement loss, cell morphology In vitro or in vivo, such as the acquisition or loss of immortalization, the acquisition or loss of tumor-specific markers, the ability to form or suppress tumors when injected into a suitable animal host, and / or the immortalization of cells It means any change in the characteristics of cell growth and proliferation. For example, Freshney, “Culture of Animal Cells, Manual of Basic Technique”, pages 231-241 (3rd edition, 1994).
[0100]
“Tumor cell” means precancerous, cancerous, and normal cells in a tumor.
[0101]
“Malignant transformation” in a “cancer cell”, “malignant transformed” cell or tissue culture means a naturally occurring or induced phenotypic change that is not necessarily related to the uptake of new genetic material. Malignant transformation results from infection with the transforming virus and integration of new genomic DNA, or uptake of foreign DNA, which occurs either spontaneously or after exposure to carcinogens, thereby causing endogenous Genes can be mutated. Malignant transformation is associated with phenotypic changes, such as cell immortalization, abnormal growth control, non-morphological changes, and / or malignancies (eg, Freshney, “Animal cell culture, Basic Manual (Culture of Animal Cells a Manual of Basic Technique), (3rd edition, 1994)).
[0102]
“Antibody” means a polypeptide comprising a framework region of an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the myriad immunoglobulin variable region genes in addition to the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Typically, the antigen-binding region of an antibody or functional equivalent thereof is most important in binding specificity and affinity. See Paul's "Fundamental Immunology".
[0103]
An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of the same pair of two polypeptide chains, each pair having one “light” chain (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that contributes primarily to antigen recognition. These terms variable light chain (V_L) And variable heavy chain (V_H) Means these light and heavy chains respectively.
[0104]
Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests the antibody under the disulfide bond in the hinge region, and F (ab) '₂Which forms a Fab dimer, which is itself V through a disulfide bond._H-C_H1 is a light chain linked to 1. This F (ab) ’₂Is reduced under moderate conditions and breaks the disulfide bond in the hinge region, thereby F (ab) '₂The dimer is converted to Fab 'monomer. Fab 'monomers are Fabs with essentially a part of the hinge region (see "Basic Immunology" (Paul, 3rd edition, 1993)). Although various antibody fragments are defined with respect to digestion of complete antibodies, those skilled in the art will recognize that such fragments can be synthesized de novo, either chemically or using recombinant DNA techniques. Will understand. Thus, the term antibody as used herein uses antibody fragments (eg, single chain Fv) or phage display libraries made by modification of whole antibodies or newly synthesized using recombinant DNA techniques. Also includes any of the identified antibody fragments (see, eg, McCafferty et al., Nature, 348: 552-554 (1990)).
[0105]
Many techniques known in the art can be used for the preparation of antibodies, eg, recombinant, monoclonal, or polyclonal antibodies (eg, Kohler and Milstein, Nature, 256: 495-497 (1975)). Kozbor et al., Immunology Today 4:72 (1983); Cole et al., Pp. 77-96, Monoclonal Antibodies and Cancer Therapy (1985); Coligan, Current Protocols in Immunology, 19A; (1988); and Goding, Monoclonal Antibodies: P rinciples and Practice (2nd edition, 1986)). Techniques for generating single chain antibodies (US Pat. No. 4,946,778) can be applied to generate antibodies to the polypeptides of the present invention. In addition, other organisms such as transgenic mice or other mammals can be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to a selected antigen (eg, McCafferty et al., Nature, 348: 552-554 (1990); Marks). Et al., Biotechnology, 10: 779-783 (1992)).
[0106]
A “chimeric antibody” is a class, effector function and / or species constant region in which (a) the constant region, or part thereof, is altered, substituted or exchanged and the antigen binding site (variable region) is different or altered, Or the chimeric antibody is linked to a different molecule in its entirety that confer new properties such as enzymes, toxins, hormones, growth factors, drugs, etc .; or (b) the variable regions, or parts thereof, are different or Antibody molecules that are altered, substituted or exchanged by variable regions with altered antigen specificity.
[0107]
Identification of sequences associated with prostate cancer
In certain aspects, the level of gene expression is determined to provide an expression profile in various patient samples where diagnostic information is desired. The expression profile of a particular sample is essentially a “fingerprint” of the state of the sample; the two states have any particular gene expressed in the same way, but evaluation of many genes Allows simultaneous generation of gene expression profiles characterizing the condition. That is, normal tissue (eg, normal prostate or other tissue) can be distinguished from prostate cancerous or metastatic cancerous tissue, or prostate cancer tissue or metastatic prostate cancerous tissue is alive It can be compared with prostate tissue samples from cancer patients and other tissues. Comparing tissue expression profiles in various known prostate cancer states gives information on which genes are important in each of these states, including both up-regulation and down-regulation of genes .
[0108]
The identification of differentially expressed sequences in prostate cancer versus non-prostate cancer tissue allows the use of this information in a number of ways. For example, a particular treatment modality can be evaluated: a chemotherapeutic agent acts to down-regulate prostate cancer in a particular patient and thus tumor growth or recurrence. Similarly, diagnostic and therapeutic outcomes can be made or confirmed by comparison with a known expression profile of a patient sample. Metastatic tissue can also be analyzed to determine the prostate cancer stage of the tissue. Furthermore, these gene expression profiles (or individual genes) allow screening of drug candidates while looking at mimicking or altering specific expression profiles; for example, screening can be drugs that suppress prostate cancer expression profiles Can be done about. This can be done by creating a biochip containing a significant set of prostate cancer genes that can then be used in these screens. These methods can also be performed on a protein basis; that is, the protein expression level of prostate cancer protein can be assessed for diagnostic purposes or to screen candidate substances. In addition, prostate cancer nucleic acid sequences can be administered for gene therapy purposes, including administration of antisense nucleic acids or prostate cancer proteins (including antibodies and other modulators) that are administered as therapeutic agents.
[0109]
The present invention thus provides nucleic acid and protein sequences that are differentially expressed in prostate cancer, referred to herein as “prostate cancer sequences”. As outlined below, prostate cancer sequences are down-regulated (ie, expressed at a lower level) in addition to sequences that are up-regulated (ie, expressed at a higher level) in prostate cancer. including. In preferred embodiments, the prostate cancer sequences are derived from humans; however, one of skill in the art will appreciate that other living prostate cancer sequences are also useful in animal models of disease and drug evaluation. Therefore other prostate cancer sequences are present in rodents (rats, mice, hamsters, guinea pigs, etc.), primates, livestock (sheep, goats, pigs, cows, horses, etc.) and pets (dogs, cats, etc.); Provided from vertebrates, including mammals. Other living prostate cancer sequences can be obtained using the techniques outlined below.
[0110]
Prostate cancer sequences include both nucleic acid and amino acid sequences. As understood by those skilled in the art and outlined in more detail below, prostate cancer nucleic acid sequences are useful for a variety of applications, including diagnostic applications that detect natural nucleic acids as well as screening applications; Biochips can be made that contain PCR microtiter plates with nucleic acid probes or probes for selected prostate cancer sequences.
[0111]
Prostate cancer sequences can be initially identified by substantial nucleic acid and / or amino acid sequence homology to the prostate cancer sequences outlined herein. Such homology can be based on the entire nucleic acid or amino acid sequence and is generally determined as outlined below, using either a homology program or hybridization conditions.
[0112]
To identify sequences associated with prostate cancer, prostate cancer screening typically involves, for example, normal and cancerous tissues, or tumor tissue samples from patients with metastatic disease, versus non-metastatic Includes comparison of genes identified in different tissues, such as tissues. Another suitable tissue comparison includes comparison of prostate cancer samples with metastatic cancer samples of other cancers such as lung cancer, breast cancer, digestive cancer, ovary, and the like. Different stages of prostate cancer, such as living tissue, drug resistance, and metastatic tissue, are applied to biochips containing nucleic acid probes. These samples, if applicable, are first microdissected and processed as is known in the art for mRNA preparation. Suitable biochips are commercially available, for example from Affymetrix. A gene expression profile as described herein is created and the data is analyzed.
[0113]
In certain embodiments, the gene that exhibits altered expression during normal and disease states is preferably normal prostate in other normal tissues, but is lung, heart, brain, liver, breast, kidney, muscle, colon, small intestine Compared to genes expressed in, including, but not limited to, large intestine, spleen, bone and placenta. In preferred embodiments, genes identified during prostate cancer screening that are expressed in significant amounts in other tissues are removed from this profile, but in some embodiments this is not necessary. That is, when a drug is screened, it is preferred that the target be disease specific in order to minimize possible side effects that are usually possible.
[0114]
In preferred embodiments, prostate cancer sequences are upregulated in prostate cancer; that is, the expression of these genes is higher in prostate cancer tissue than in non-cancerous tissue. “Up-regulation” as used herein often means at least about 2-fold change, preferably at least about 3-fold change, and preferably at least about 5-fold or more. All unigene cluster identification numbers and accession numbers herein are relative to the GenBank sequence database, and the sequence of accession numbers is expressly incorporated herein by reference. GenBank is known in the art and is described, for example, in Benson, DA et al., Nucleic Acids Research, 26: 1-7 (1998) and http: // www. ncbi. nlm. nih. See gov /. The sequences are also available in other databases, examples being the European Molecular Biology Laboratory (EMBL) and the Japanese DNA database (DDBJ).
[0115]
In another preferred embodiment, the prostate cancer sequence is one that is down-regulated in prostate cancer; that is, the expression of these genes is lower in prostate cancer tissue compared to non-cancerous tissue (eg, Tables 8, 12). And 14). “Down-regulation” as used herein often means at least about a 1.5-fold change, more preferably a 2-fold change, preferably at least about 3-fold change, and Most preferred is a change of about 5 times or more.
[0116]
Informatics
The ability to identify genes that are over- or under-expressed in prostate cancer is high in diagnostic, therapeutic, drug development, pharmacogenetics, protein structure, biosensor development, and other related fields. A database with high resolution and high sensitivity can be additionally provided. For example, the expression profile can be used for diagnosis or prognosis assessment of prostate cancer patients. Otherwise, as another example, subcellular toxicity information can be generated to better indicate the correlation between drug structure and activity (Anderson, “Pharmaceutical Proteomics: Targets, Mechanisms, and Functions (Pharmacological) Proteomics: Targets, Mechanism, and Function) ", IBC Proteomics Conference, Coronado, CA (June 11-12, 1998)). Subcellular level toxicity information can also be used in biological sensor devices, perhaps to estimate the toxic effects of chemical exposure and possible tolerable exposure thresholds (US Pat. No. 5,811,11). 231). Similar benefits are generated from databases on other biomolecules and bioactive agents (eg, nucleic acids, sugars, lipids, drugs, etc.).
[0117]
Accordingly, in another aspect, the present invention provides a database comprising at least one set of assay data. The data contained in this database is obtained, for example, using array analysis, either alone or in a library format. This database can be in virtually any form in which data is maintained and communicated, but is preferably an electronic database. The electronic database of the present invention can be maintained on an electronic device that allows storage and access to the database, such as a personal computer, but can be distributed to a wide area network such as the World Wide Web (WWW). preferable.
[0118]
This section on databases containing peptide sequence data focuses only on clarity of explanation. One skilled in the art will also appreciate that similar databases can be compiled for assay data acquired using the assay methods of the present invention.
[0119]
Identification and / or quantification of a wide variety of molecular and macromolecular species, relative to and / or absolutely abundant, from biological samples that are prostate cancer, ie, sequences related to prostate cancer as described herein The composition and method for identifying abundant information provides a wealth of information, including the determination of pathology, disease predisposition, drug testing, treatment monitoring, causal linkages to genetic diseases, correlations with immune and physiological conditions Etc. Although the data generated from the assay of the present invention is suitable for mammalian validation and analysis, in a preferred embodiment, prior data being processed using a high speed computer is utilized.
[0120]
A series of methods for indexing and retrieving biomolecular information is known in the art. For example, US Pat. Nos. 6,023,659 and 5,966,712 describe biomolecular sequence information in a manner that allows sequences to be cataloged and searched according to a hierarchy of one or more protein functions. A relational database system for storing data is disclosed. US Pat. No. 5,953,727 collects partial length DNA sequences that are cataloged and searched according to one or more sequencing projects to obtain a full length sequence from the collection of partial length sequences. Discloses a relational database having sequence records containing information in a format that enables US Pat. No. 5,706,498 discloses a gene for searching for similar gene sequences in sequence data items in a gene database based on the degree of similarity between a key sequence and a target sequence. A database search system is disclosed. US Pat. No. 5,538,897 is for identifying amino acid sequences in computer databases by comparing estimated mass spectra with empirically derived mass spectra using a closeness-of-fit measurement. Discloses a method for using mass spectrometry fragmentation patterns of peptides. US Pat. No. 5,926,818 describes multidimensional data described as online analysis processing (OLAP), which entails the integration of actual data estimated according to more than one integration path or dimensions. A multi-dimensional database including analytical correlation is disclosed. U.S. Pat. No. 5,295,261 discloses a hybrid database structure in which the fields of each database record are divided into two classes, namely navigation data and informational data, the navigation field being a dendritic structure. Alternatively, it is stored in the form of a hierarchical topological map that can be viewed as the union of two or more such dendritic structures.
[0121]
See also Mount et al., Bioinformatics (2001); “Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids”, Ed. Bioinformatics: A practical guide to gene and protein analysis (Bioinformatics: The Analysis of Genes and Proteins) (Baxevanis and Oeulette, Ed., Biobiology, Biomedical and Bioinformatics, Bioinformatics in Biomedics, Bioinformatics and Bioinformatics in Biomedics) Meet “Bioinformatics: Basic Applications in Biological Science and Medicine” (1999); “Introduction to Computational Molecular Biology” (Ed. : Methods and Protocols "(Misser and Krawetz, 2000);" Bionformatics: Sequences, Structures and Databanks: Practical Methods (Bioinformatics: Sequences, Structures and Databanks: A Practical). " (Prog) (Edited by Higgins and Taylor, 2000); Brown, “Bioinformatics: A Biologics' Guide to Biocomputing and the Internet” (2001); Han; "Data Mining: Concepts and Techniques" (2000); and Waterman, "Introduction of Computer Biology: Maps, Sequences and Computational Biology: Maps, Sequences and Sequences, Sequences and Sequences." (1995) Of it.
[0122]
The present invention includes, for example, a computer and software for storing assay data records in a form that can be searched by a cross-tabulated computer based on data specifying a source of a target-containing sample from which a specificity record of each sequence is obtained. A computer database including
[0123]
In an exemplary embodiment, the source of the at least one target-containing sample is derived from a control tissue sample that is known to be independent of pathological disease. In a variant, the at least one source is a known pathological tissue specimen, such as another tissue specimen to be analyzed for neoplastic lesions or prostate cancer. In another variation, the assay record cross-tabulates one or more of the following parameters for each target specimen in the sample: (1) eg target molecular structure coordinates and / or characteristic separation coordinates (eg electrophoretic coordinates) ) A unique identification code; (2) a sample source; and (3) an absolute and / or relative amount of target species present in the sample.
[0124]
The present invention is further provided for storage and retrieval of target data collection in a computer data storage device, which is a magnetic disk, optical disk, magneto-optical disk, DRAM, SRAM, SGRAM, SDRAM, RDRAM, DDRRAM. , Magnetic bubble memory devices, and other data storage devices including CPU registers and on-CPU data storage arrays. Typically, target data recording is performed as a bit pattern in an array of magnetic domains on a magnetizable medium or as an array of charge states or transistor gate states, eg, an array of cells in a DRAM device (eg, each cell is a transistor, And a storage region that can be located on the transistor). In one aspect, the present invention provides such a storage device, and a computer system built therebetween, which includes a unique identifier for at least 10 target data records cross-tabulated with the target source. It includes a bit pattern that encodes a protein expression fingerprint record.
[0125]
Where the target is a peptide or nucleic acid, the present invention preferably provides a method of identifying the sequence of the relevant peptide or nucleic acid, which is stored or retrieved from a computer storage device or database. Performing a computerized comparison between the sequence assay record and at least one other sequence. The comparison can include a sequence analysis or comparison algorithm or a computer program as an aspect thereof (eg, FASTA, TFASTA, GAP, BESTFIT) and / or the comparison is determined from a polypeptide or nucleic acid sample of the specimen. It can be the relative amount of peptide or nucleic acid sequence in the pool of sequences rendered.
[0126]
The present invention includes an IBM compatible (DOS, Windows) bit pattern encoding the assay data of the present invention in a file format suitable for computer input sequence analysis, comparison, or search and processing in relative quantification methods. , Windows 95/98/2000, Windows NT, OS / 2) or other formats (eg Linux, SunOS, Solaris, AIX, SCO Unix, VMS, MV, Macintosh, etc.) floppy diskette or hard (built-in, Winchester) disk It is also preferred to provide a magnetic disk, such as a drive.
[0127]
The present invention further comprises a plurality of computing devices connected via a data link such as an Ethernet cable (coaxial or 10BaseT), telephone line, ISDN line, wireless network, optical cable, or other suitable signal transmission medium. Also provides a network whereby at least one network device (eg, computer, disk array, etc.) constitutes a bit pattern encoding data obtained from an assay of the present invention, comprising a magnetic domain ( For example, a magnetic disk) and / or a charge domain (eg, an array of DRAM cells).
[0128]
The present invention also provides a method for transmitting assay data comprising generating an electronic signal on an electronic communication device such as a modem, ISDN terminal adapter, DSL, cable modem, ATM switch, wherein the signal is ( It includes a bit pattern encoding a database containing data from the assay (in native or encrypted format) or multiple assay results obtained by the method of the invention.
[0129]
In a preferred embodiment, the present invention compares the query target to a database containing an array of data structures, such as assay results obtained by the method of the present invention, and is based on the identity of the target data and the degree of gap weight. A computer system for ranking database targets is provided. The central processing unit is initialized and a computer program for juxtaposition and / or comparison of assay results is read and executed. The inquiry target data is input to the central processing unit via the I / O device. Execution of the computer program causes the central processing unit to retrieve assay data from the data file, which includes a binary description of the assay results.
[0130]
Target data or records and computer programs can be transferred to secondary memory, which is typically random access memory (eg, DRAM, SRAM, SGRAM, or SDRAM). The targets are ranked according to the selected assay characteristic (eg, binding to the selected affinity moiety) and the degree of matching of the same characteristic with the query target, and the result is output via the I / O device. For example, the central processing unit can be a normal computer (eg, Intel Pentium, PowerPC, Alpha, PA-8000, SPARC, MIPS 4400, MIPS 10000, VAX, etc.); It can be a molecular biology software package (eg UWGCG Sequence Analysis Software, Darwin); data files are optical or magnetic disks, data servers, memory devices (eg DRAM, SRAM, SGRAM, SDRAM, EPROM, bubble memory) I / O devices include video displays and keyboards, modems, ISDN terminal adapters, Ethernet Port labeled, punch card reader can be a magnetic strip reader or other suitable I / O terminals with a device.
[0131]
The present invention also preferably provides for the use of a computer system as described above, which includes: (1) a computer; (2) obtained by the method of the present invention, which can be stored in a computer. A conserved bit pattern encoding a collection of specific peptide sequence specificity records; (3) a comparison target, such as a query target; and (4) typically based on computerized similarity values This is a program for juxtaposition and comparison by rank-ordering of comparison results.
[0132]
Characteristics of proteins related to prostate cancer
The prostate cancer proteins of the present invention can be classified as secreted proteins, transmembrane proteins or intracellular proteins. In certain embodiments, the prostate cancer protein is an intracellular protein. Intracellular proteins can be found in the cytoplasm and / or nucleus. Intracellular proteins are involved in all aspects of cellular function and replication, including signal transduction pathways; aberrant expression of such proteins often results in unregulated or unregulated cellular processes (eg, , "Molecular Biology of the Cell" (Alberts, 3rd edition, 1994)). Many intracellular proteins have enzyme activities such as protein kinase activity, protein phosphorylation activity, protease activity, nucleotide cyclase activity, and polymerase activity. Intracellular proteins are also used as docking proteins that are involved in organizing protein complexes or targeting proteins to various subcellular locations and in maintaining the structural integrity of organelles Can do.
[0133]
An increasingly understood concept in protein characterization is the presence of one or more motif proteins to which a defined function contributes. In addition to the highly conserved sequences found in the enzyme domains of proteins, highly conserved sequences are identified in proteins associated with protein-protein interactions. For example, the Src-homology 2 (SH2) domain binds to tyrosine phosphorylated targets in a sequence-dependent manner. A PTB domain, which is distinct from the SH2 domain, also binds to tyrosine phosphorylated targets. The SH3 domain binds to a proline rich target. In addition, only a few named PH domains, tetratricopeptide repeats and WD domains have been shown to mediate protein-protein interactions. Some of these are also associated with binding to phospholipids or other second messengers. As will be appreciated by those skilled in the art, these motifs can be identified on the basis of the primary sequence; thus, analysis of protein sequences can be performed enzymatically for both molecules and / or molecules that can associate with proteins. Can provide insight into sex. One useful database is Pfam (protein family), which is a large collection of hidden Markov models that cover multiple sequence alignments and many common protein domains. Versions are available on the Internet from the University of Washington (St. Louis), the Sanger Center (UK), and the Karolinska Institute (Sweden) (eg, Bateman et al., Nuc. Acids Res., 28: 263-266 (2000); Sonhammer et al., Proteins, 28: 405-420 (1997); Bateman et al., Nuc. Acids Res., 27: 260-262 (1999); Et al., Nuc. Acids Res., 26: 320-322 (1998)).
[0134]
In another embodiment, the prostate cancer sequence is a transmembrane protein. A transmembrane protein is a molecule that spans the phospholipid bilayer of a cell. These have an intracellular domain, an extracellular domain, or both. The intracellular domain of such a protein can have many functions, including those already described for intracellular proteins. For example, the intracellular domain has enzymatic activity and / or can be utilized as a binding site for additional proteins. The intracellular domain of transmembrane proteins often plays both roles. For example, some receptor tyrosine kinases have both protein kinase activity and SH2 domains. In addition, autophosphorylation of the tyrosine receptor molecule itself forms a binding site for proteins that contain additional SH2 domains.
[0135]
A transmembrane protein can comprise one to many transmembrane domains. For example, receptor tyrosine kinases, certain cytokine receptors, receptor guanylyl cyclase and receptor serine / threonine protein kinases contain one transmembrane domain. However, various other proteins including channels and adenylyl cyclase contain multiple transmembrane domains. Many important cell surface receptors, such as G protein-coupled receptors (GPCRs), contain a region that spans seven membranes, so they are classified as “seven transmembrane domains” proteins. The characteristics of the transmembrane domain include approximately 20 consecutive hydrophobic amino acids followed by charged amino acids. Thus, when analyzing the amino acid sequence of a particular protein, the localization and number of transmembrane domains within the protein can be estimated (see, eg, PSORT website, http://psort.nibb.ac.jp/). . Important transmembrane protein receptors are insulin receptor, insulin-like growth factor receptor, human growth hormone receptor, glucose transporter, transferrin receptor, epidermal growth factor receptor, low density lipoprotein receptor, epidermal growth factor Receptors, leptin receptors, interleukin receptors such as, but not limited to, IL-1 receptor, IL-2 receptor and the like.
[0136]
The extracellular domains of transmembrane proteins are diverse; however, conserved motifs are found repeatedly between the various extracellular domains. Conserved structures and / or functions are due to different extracellular motifs. Many extracellular domains are associated with binding to other molecules. In certain aspects, the extracellular domain is found on the receptor. Factors bound to the receptor domain include circulating ligands, which can be small molecules such as peptides, proteins or adenosine. For example, growth factors such as EGF, FGF and PDGF are circulating growth factors that bind to their cognate receptors and initiate various cellular responses. Other factors include cytokines, mitogenic factors, neurotrophic factors and the like. The extracellular domain also binds to molecules associated with the cell. In this respect, they mediate cell-cell interactions. The ligands associated with the cells can be bound to the cells, eg, by glycosyl phosphatidylinositol (GPI) anchors, or they can themselves be transmembrane proteins. The extracellular domain is also associated with the extracellular matrix and contributes to the maintenance of the cell structure.
[0137]
These are particularly preferred in the present invention because transmembrane prostate cancer proteins are readily accessible targets for immunotherapy, as described herein. In addition, as outlined below, transmembrane proteins are also useful in imaging modalities. Antibodies can be used to label such readily accessible proteins in situ. Alternatively, the antibody can label intracellular proteins, in which case the sample typically increases permeability to gain access to the intracellular proteins.
[0138]
It will also be appreciated by those skilled in the art that transmembrane proteins can be made soluble by removing transmembrane sequences, for example, by recombinant methods. Furthermore, the transmembrane protein rendered soluble can be secreted through recombinant means by adding an appropriate signal sequence.
[0139]
In another embodiment, the prostate cancer protein is a secreted protein; its secretion can be either constitutive or regulated. These proteins contain a signal peptide or signal sequence that targets this molecule to the secretory pathway. Secreted proteins are associated with many physiological events; due to their circulating nature, they are utilized for signaling to various other cell types. This secreted protein acts in the autocrine mode (acting on cells secreting the factor), the paracrine mode (acting on cells very close to the cell secreting the factor) or the endocrine mode (acting on distant cells) ). Thus, secreted molecules find use in modulating or altering many physiological aspects. Prostate cancer protein, a secreted protein, is particularly preferred in the present invention because it is used as a good target for diagnostic markers, such as blood, plasma, serum or stool tests.
[0140]
Use of prostate cancer nucleic acid
As mentioned above, prostate cancer sequences are initially identified by substantial nucleic acid and / or amino acid sequence homology or linkage with the prostate cancer sequences outlined herein. Such homology can be based on the overall nucleic acid or amino acid sequence and is generally determined using either a homology program or hybridization conditions, as outlined below. Typically, sequences linked on mRNA are found on the same molecule.
[0141]
The prostate cancer nucleic acid sequences of the present invention, such as the sequences of Tables 1-16, can be relatively large gene fragments, ie, they are nucleic acid segments. “Gene” in this context includes coding regions, non-coding regions, and a mixture of coding and non-coding regions. Thus, as will be appreciated by those skilled in the art, using the sequences provided herein, the sequence extended in either direction of the prostate cancer gene, either a relatively long sequence or a full length sequence is cloned. Can be obtained using techniques well known in the art. See the article by Ausubel et al. (Supra). Many can be done by informatics, and many sequences are clustered to contain multiple sequences corresponding to a single gene, such as a system like UniGene (http: //www.ncbi. nlm.nih.gov/UniGene/see).
[0142]
Once the prostate cancer nucleic acid has been identified, it can be cloned, and if necessary, its constitutive parts are recombined to form the entire prostate cancer nucleic acid coding region or the entire mRNA sequence. Once isolated from its natural source and contained in, for example, a plasmid or other vector, or excised therefrom as a linear nucleic acid segment, the recombinant prostate cancer nucleic acid can be used as a probe, eg, an expanded code. Like the region, it can be further used to identify and isolate other prostate cancer nucleic acids. It can also be used to make modified or variant prostate cancer nucleic acids and proteins as “precursor” nucleic acids.
[0143]
The prostate cancer nucleic acids of the invention are used in several ways. In a first aspect, as outlined below, a nucleic acid probe for prostate cancer nucleic acid is made and attached to a biochip used in screening and diagnostic methods, or for example gene therapy, vaccine and / or anti-antigen Administered for sense use. Alternatively, a prostate cancer nucleic acid comprising a prostate cancer protein coding region can be incorporated into an expression vector for expression of the prostate cancer protein, and for screening purposes or for administration to a patient.
[0144]
In a preferred embodiment, nucleic acid probes (both the nucleic acid sequences outlined in the figure and / or their complements) are made to prostate cancer nucleic acids. The nucleic acid probe bound to the biochip is designed to be substantially complementary to the prostate cancer nucleic acid, ie, the target sequence (eg, either the target sequence of the sample or other probe sequence in a sandwich assay). As a result, hybridization between the target sequence and the probe of the present invention occurs. As outlined below, this need for complementarity is not perfect; even if there are several base pair mismatches that would interfere with hybridization between the target sequence and the single stranded nucleic acid of the invention. Good. However, if the number of mutations is too large, hybridization will not occur even if the hybridization conditions are minimal stringent, and this sequence is not a complementary target sequence. Thus, “substantial complementarity” herein is sufficiently complementary to allow the probe to hybridize to the target sequence under normal reaction conditions, particularly under high stringency conditions, as outlined below. Means sex.
[0145]
The nucleic acid probe is generally single-stranded, but may be partially single-stranded and partially double-stranded. The strand formation of this probe is specified by the structure, composition, and properties of the target sequence. In general, nucleic acid probes range from about 8 to about 100 bases in length, with about 10 to about 80 bases being preferred, and about 30 to about 50 bases being particularly preferred. That is, generally all genes are not used. In some embodiments, very long nucleic acids can be used, ranging up to several hundred bases.
[0146]
In a preferred embodiment, more than one probe per sequence is used, either overlapping probes or probes to different compartments of the target used. That is, 2, 3, 4 or more probes, preferably 3 are used to construct the redundancy of a particular target. These probes can overlap (ie have some sequences in common) or be separated. In some cases, PCR primers can be used to amplify a more sensitive signal.
[0147]
As will be appreciated by those skilled in the art, nucleic acids can be bound or immobilized to a solid support in a wide variety of ways. As used herein, “fixed” and grammatical equivalents refer to the association or binding between a nucleic acid probe and a solid support, and the solid support is a binding, as outlined below, It is sufficient to be stable under the conditions of washing, analysis and removal. This binding can typically be covalent or non-covalent. As used herein, “non-covalent bonds” and grammatical equivalents refer to one or more electrostatic, hydrophilic and hydrophobic interactions. Included in the non-covalent bond is a covalent attachment of the molecule, eg, to a streptavidin support, and a non-covalent bond of a biotinylated probe to streptavidin. As used herein, “covalent bond” and grammatical equivalent means that the two parts of the solid support and the probe are attached by at least one bond, which is a σ bond, a π bond and Includes coordination bonds. Covalent bonds can be formed directly between the probe and the solid support, or specific reactive groups cross-linked or inclusion either on the solid support or on the probe or on both molecules. Can be formed. Immobilization can also involve a combination of covalent and non-covalent interactions.
[0148]
In general, these probes can be attached to the biochip in a wide variety of ways, as will be appreciated by those skilled in the art. As noted herein, nucleic acids may be synthesized first and then attached to the biochip or synthesized directly on the biochip.
[0149]
The biochip includes a suitable solid phase substrate. As used herein, a “substrate” or “solid support” or other grammatical equivalent can be modified to include individual individual sites suitable for attachment or association of nucleic acid probes, at least one It means a material that can be examined based on the detection method of the species. As will be appreciated by those skilled in the art, the number of possible substrates is very large, as well as glass and modified or functionalized glass, plastic (acrylic, polystyrene, and copolymers of styrene and other materials, Polypropylene, polyethylene, polybutylene, polyurethane, TeflonJ, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silicone and modified silicones, carbon, metals, inorganic glasses, silica-based materials including plastics, etc. However, it is not limited to these. In general, these substrates are optically detectable and do not fluoresce so that they can be recognized. A preferred substrate is US patent application Ser. No. 09 / 270,214, filed Mar. 15, 1999, entitled “Reusable Low Fluorescent Plastic Biochip”. Which is incorporated herein by reference.
[0150]
Generally, the substrate is a flat plate, but other substrate shapes may be used as will be appreciated by those skilled in the art. For example, these probes can be placed on the inner surface of the tube for sample analysis flow-through to minimize sample volume. Similarly, these substrates can be flexible like flexible foam, including closed cells made of certain plastics.
[0151]
In a preferred embodiment, the biochip and probe surfaces can be derivatized with chemical functional groups for these two subsequent attachments. Thus, for example, a biochip can be derivatized with chemical functional groups such as, but not limited to, amino groups, carboxyl groups, oxo groups and thiol groups, with amino groups being particularly preferred. By using these functional groups, the probe is attached using functional groups on the probe. For example, nucleic acids containing amino groups can be attached to surfaces containing amino groups, for example using linkers known in the art; for example, homobifunctional or heterobifunctional linkers are also well known ( (Ref. 1994, Pierce Chemical Catalog, Cross-Linker Technology, pp. 155-200). In addition, in some cases, additional linkers such as alkyl groups (including substituted and heteroalkyl groups) can be used.
[0152]
In this embodiment, the oligonucleotide is synthesized as is known in the art and then attached to the surface of the solid support. As will be appreciated by those skilled in the art, either the 5 'or 3' end may be attached to the solid support or the attachment may be via an internal nucleoside.
[0153]
In another embodiment, immobilization to a solid support is very strong but still non-covalent. For example, biotinylated oligonucleotides can be made that bind to a surface that is covalently coated with streptavidin, resulting in attachment.
[0154]
Alternatively, oligonucleotides can be synthesized on the surface, as is known in the art. For example, photoactivation techniques using photopolymerizable compounds and techniques are used. In a preferred embodiment, these nucleic acids are well known, such as WO 95/25116; WO 95/35505; US Pat. Nos. 5,700,637 and 5,445,934. Can be synthesized in situ using photolithographic techniques; and all references cited therein are expressly incorporated herein by reference; these attachment formation methods are based on the Affimetrix GeneChip ™ technology. That forms the basis of
[0155]
Often, amplification-based assays are performed to measure the level of expression of sequences associated with prostate cancer. These assays are typically performed in combination with reverse transcription. In such assays, nucleic acid sequences associated with prostate cancer serve as templates in amplification reactions (eg, polymerase chain reaction, or PCR). In quantitative amplification, the amount of amplification product appears to be proportional to the amount of template in the original sample. Comparison with the appropriate control provides a measure of the amount of RNA associated with prostate cancer. Methods of quantitative amplification are well known to those skilled in the art. See, for example, Innis et al., “PCR Protocols, A Guide to Methods and Applications” (1990), for detailed protocols for quantitative PCR.
[0156]
In some embodiments, TaqMan based assays are used to measure expression. TaqMan-based assays use fluorescent oligonucleotide probes that contain a fluorescent dye on the 5 'side and a quencher on the 3' side. This probe hybridizes to the PCR product but cannot itself extend due to the blocking agent at the 3 'end. If the PCR product is amplified in a continuous cycle, the 5 'nuclease activity of the polymerase, eg AmpliTaq, results in cleavage of the TaqMan probe. This cleavage separates the 5 'fluorescent dye and the 3' quencher, thereby resulting in an increase in fluorescence as a function of amplification (eg, literature provided by Perkin-Elmer, www2.perkin-elmer). .Com).
[0157]
Other suitable amplification methods include ligase chain reaction (LCR) (Wu and Wallace, Genomics 4: 560 (1989), Landegren et al., Science 241: 1077 (1988), and Barringer et al., Gene 89: 117 ( 1990)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA, 86: 1173 (1989)), self-sustaining sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA, 87: 1874 ( 1990)), dot PCR, linker adapter PCR, and the like, but are not limited thereto.
[0158]
Expression of prostate cancer protein from nucleic acids
In a preferred embodiment, a prostate cancer nucleic acid encoding, for example, a prostate cancer protein is used to generate various expression vectors for expression of the prostate cancer protein and subsequent use in screening assays, as described below. Expression vectors and recombinant DNA techniques are well known to those skilled in the art (see, eg, Ausubel, supra, and “Gene Expression Systems” (Fernandez and Hoeffler, 1999)) and express proteins. Can be used for. Expression vectors can be either self-replicating extrachromosomal vectors or vectors that integrate into the host genome. In general, these expression vectors comprise transcriptional and translational regulatory nucleic acids operably linked to nucleic acid encoding prostate cancer protein. The term “control sequence” refers to a DNA sequence that is used to express a coding sequence operably linked in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
[0159]
A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, a DNA for a presequence or secretion leader is operably linked to the DNA of the polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer can transcribe the sequence. Is operably linked to the coding sequence; or the ribosome binding site is operably linked to the coding sequence if it is positioned to facilitate translation. In general, “operably linked” means that the linked DNA sequences are contiguous, as well as in the reading phase in the case of a secretory leader. However, enhancers do not have to be contiguous. Ligation is typically accomplished by convenient ligation of restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with normal practice. Transcriptional and translational regulatory nucleic acids are generally suitable for host cells used to express prostate cancer proteins. Many types of suitable expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
[0160]
In general, transcriptional and translational regulatory sequences can include, but are not limited to, promoter sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. In a preferred embodiment, the regulatory sequences include a promoter and transcription start and stop sequences.
[0161]
A promoter sequence encodes either a constitutive or inducible promoter. These promoters are either natural promoters or hybrid promoters. Hybrid promoters combine more than one promoter element, which are also known in the art and useful in the present invention.
[0162]
In addition, the expression vector can include additional elements. For example, an expression vector may have two replication systems and is therefore maintained in two organisms, such as in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. Can. Furthermore, in order to incorporate an expression vector, the expression vector has at least one sequence homology to the host cell genome and preferably has two homologous sequences flanking the expression construct. The integrating vector can be directed to a specific locus in the host cell by selection of appropriate homologous sequences for inclusion within the vector. Constructs for vector integration are well known in the art (eg, Fernandez and Hoeffler, supra).
[0163]
In addition, in a preferred embodiment, the expression vector contains a selectable marker gene that allows selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.
[0164]
The prostate cancer protein of the present invention is obtained by culturing host cells transformed with an expression vector containing a nucleic acid encoding prostate cancer protein under conditions suitable for inducing or inducing expression of the prostate cancer protein. Created. Suitable conditions for prostate cancer protein expression will vary with the choice of the expression vector and the host cell, and those skilled in the art will readily ascertain this through routine experimentation or optimization. For example, the use of constitutive promoters in expression vectors is necessary to optimize host cell growth and proliferation, while the use of inducible promoters requires appropriate growth conditions for induction. . In addition, in some embodiments, the timing of recovery is important. For example, the baculovirus system used for insect cell expression is a lytic virus so that the choice of harvest time is critical to product yield.
[0165]
Suitable host cells are yeast, bacteria, archaea, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, Sf9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, CO, HUVEC (human umbilical vein epithelial cells), THP1 cells (macrophage cell lines) and various other human cells and cell lines.
[0166]
In a preferred embodiment, the prostate cancer protein is expressed in mammalian cells. Mammalian expression systems are also known in the art and include retroviral and adenoviral systems. One expression vector system is a retroviral vector system, as generally disclosed in PCT / US97 / 01019 and PCT / US97 / 01048, both of which are hereby expressly incorporated by reference. In particular, a promoter derived from a mammalian viral gene is used as a mammalian promoter because the viral gene is often highly expressed and has a wide host region. Examples are the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and CMV promoter (see, eg, Fernandez and Hoeffler, supra). Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3 'to translate stop codons and thus flanking the coding sequence along with the promoter element. ing. Examples of transcription terminator signals and polyadenylation signals include those derived from SV40.
[0167]
Methods for introducing exogenous nucleic acid into mammalian hosts in addition to mammalian hosts are well known in the art and will vary with the host cell used. The technology includes dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Including.
[0168]
In a preferred embodiment, the prostate cancer protein is expressed in a bacterial system. Bacterial expression systems are well known in the art. Bacteriophage derived promoters are also used and are known in the art. In addition, synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of trp and lac promoter sequences. Furthermore, bacterial promoters can include native promoters that are not of bacterial origin and have the ability to bind bacterial RNA polymerase and initiate transcription. In addition to a functional promoter sequence, an efficient ribosome binding site is desirable. This expression vector also contains a signal peptide sequence that provides for secretion of prostate cancer protein in bacteria. This protein is secreted into the growth medium (Gram positive bacteria) or into the periplasmic space located between the inner and outer membranes of the cells (Gram negative bacteria). Bacterial expression vectors can further include a selectable marker gene that allows selection of transformed bacterial species. Suitable selection genes include genes that confer bacterial resistance to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers further include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways. These components are assembled into an expression vector. Bacterial expression vectors are well known in the art and include Bacillus subtilis, E. coli, Streptococcus cremoris, Streptococcus lividans, etc. (see, for example, Fernandez and Hoeffer, supra). Bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art such as calcium chloride treatment, electroporation and the like.
[0169]
In certain embodiments, prostate cancer proteins are produced in insect cells. Expression vectors for insect cell transformation, particularly baculovirus-based expression vectors, are well known in the art.
[0170]
In a preferred embodiment, the prostate cancer protein is produced in yeast cells. Yeast expression systems are well known in the art and include Saccharomyces cerevisiae, Candida albicans and C. cerevisiae. C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. et al. K. lactis, Pichia gulerimimonii and P. lactis. Including expression vectors for P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
[0171]
Prostate cancer protein can also be produced as a fusion protein using techniques well known in the art. Thus, for example, for production of monoclonal antibodies, if the desired epitope is small, this prostate cancer protein is fused to a carrier protein to form an immunogen. Alternatively, prostate cancer protein is made as a fusion protein for increased expression or for other reasons. For example, if the prostate cancer protein is a prostate cancer peptide, the nucleic acid encoding this peptide can be linked to other nucleic acids for expression purposes.
[0172]
In a preferred embodiment, the prostate cancer protein is purified or isolated after expression. Prostate cancer protein is isolated or purified in various ways known to those skilled in the art, depending on the other components present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobicity, affinity, and reverse phase HPLC chromatography, and isoelectric focusing. For example, prostate cancer protein can be purified using a standard anti-prostate cancer protein antibody column. In conjunction with protein concentration, ultrafiltration and diafiltration techniques are also useful. For general guidance on suitable purification techniques, see “Protein Purification” (Scope) by Scopes. The degree of purification required will vary depending on the use of the prostate cancer protein. In some cases, purification may not be necessary.
[0173]
Once expressed and purified as needed, the prostate cancer proteins and nucleic acids are useful in many applications. These are useful as immunoselective reagents, vaccine reagents, screening agents and the like.
[0174]
Prostate cancer protein variants
In certain embodiments, the prostate cancer protein is a derivative or variant prostate cancer protein relative to the wild type sequence. That is, as will be described in detail below, derivative prostate cancer peptides often contain at least one amino acid substitution, deletion or insertion, with amino acid substitutions being particularly preferred. Amino acid substitutions, insertions or deletions can occur at any residue within the prostate cancer peptide.
[0175]
Further included in one embodiment of the prostate cancer protein of the present invention are amino acid sequence variants. These variants typically fall into one or more of three classes: substitutional variants, insertion variants or deletion variants. These variants are usually prepared by site directed mutagenesis of nucleotides of DNA encoding prostate cancer protein using cassette or PCR mutagenesis or other techniques well known in the art. And is then expressed in recombinant cell culture as described above. However, variant prostate cancer protein fragments having up to about 100-150 residues can be prepared by in vitro synthesis using established techniques. Amino acid sequence variants can be characterized by the predetermined nature of the variant, the ability to set them apart from the natural allele, or interspecies variation of the prostate cancer protein amino acid sequence. These variants typically exhibit the same nature of biological activity as the natural analog, but variants with modified characteristics as outlined in more detail below can also be selected.
[0176]
While the position or region for introducing an amino acid sequence variation is predetermined, the mutation itself need not be predetermined. For example, to optimize the performance of mutations at a given position, random mutagenesis can be performed at the target codon or region and the expressed prostate cancer variants screened for the optimal combination of desired activities. it can. Techniques for making substitution mutations at predetermined positions in DNA of known sequence are well known, and examples include M13 primer mutagenesis and PCR mutagenesis. Screening for these variants is performed using prostate cancer protein activity assays.
[0177]
Amino acid substitutions are typically for a single residue; insertions are usually made with about 1 to 20 digits of amino acids, but can withstand fairly large insertions. Deletions range from about 1 to about 20 residues, but in some cases may be larger deletions.
[0178]
Substitutions, deletions, insertions or any combination thereof can be used to arrive at the final derivative. In general, these changes are made in a few amino acids to minimize molecular alterations. However, relatively large changes can be tolerated in certain situations. Where minor changes in prostate cancer protein characteristics are desired, it is done according to the amino acid substitution relationships as described in the definition section.
[0179]
These variants typically display the same nature of biological activity as the natural analog and elicit the same immune response, but the variant is also chosen to modify the characteristics of the required prostate cancer protein Is done. Alternatively, this variant may be designed such that the biological activity of the prostate cancer protein is altered. For example, glycosylation sites can be altered and removed.
[0180]
Substantial changes in functional or immunological identity can be made by selecting substitutions that are less conserved than those previously described. For example, substitutions can be made that significantly affect the following: the structure of the polypeptide backbone of the modified moiety, eg, the α helix or β sheet structure; the charge or hydrophobicity of the molecule at the target site; or , Side chain bulk. In general, substitutions that are expected to result in the greatest change in the properties of the polypeptide are: (a) a hydrophilic residue such as seryl or threonyl is a hydrophobic residue such as leucyl, isoleucyl, phenylalanyl, valyl, or Being substituted by (by) alanyl; (b) cysteine or proline being substituted by (by) any other residue; (c) a residue having a positively charged side chain, eg Lysyl, arginyl or histidyl is substituted by (for) a residue having a negatively charged side chain, such as glutamyl or aspartyl; or (d) a residue having a bulky side chain, such as phenylalanine is It is to be substituted for (for) something without a chain, for example glycine.
[0181]
Covalent modifications of prostate cancer polypeptides are included within the scope of the present invention. One type of covalent modification is an organic in which a targeted amino acid residue of a prostate cancer polypeptide is capable of reacting with a selected side chain or N-terminal or C-terminal residue of the prostate cancer polypeptide. Reacting with a derivatizing agent. Derivatization with a bifunctional reagent can be performed on a water-soluble support matrix or surface of a prostate cancer polypeptide, for example, for use in a purification or screening assay for anti-prostate cancer polypeptide antibodies, as described in more detail below. It is useful for cross-linking. Commonly used crosslinking agents are, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters such as esters with 4-azidosalicylic acid such as 3,3′-dithiobis. Homobifunctional imide esters, including disuccinimidyl esters such as (succinimidyl propionate), bifunctional maleimides such as bis-N-maleimide-1,8-octane, and methyl-3- ( Reagents such as (p-azidophenyl) dithio) propioimidate are included.
[0182]
Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl, threonyl or tyrosyl residues, lysine, arginine and Methylation of amino groups of histidine side chains (Creighton, “Proteins: Structure and Molecular Properties” 79-86 (1983)), N-terminal amine acetylation, and C-terminal carboxyl group Includes amidation.
[0183]
Another type of covalent modification of prostate cancer polypeptide within the scope of the present invention involves altering the natural glycosylation pattern of the polypeptide. A “natural glycosylation pattern alteration” is for purposes herein a deletion of one or more sugar moieties found in a native sequence prostate cancer polypeptide and / or a native sequence prostate. It is intended to mean the addition of one or more glycosylation sites that are not present in a cancer polypeptide. The glycosylation pattern can be altered in many ways. For example, the use of different cell types to express sequences associated with prostate cancer can result in different glycosylation patterns.
[0184]
Addition of glycosylation sites on prostate cancer polypeptides can also be achieved by altering their amino acid sequence. This alteration can be made, for example, by the addition or substitution of a native sequence prostate cancer polypeptide with one or more serine or threonine residues (O-linked glycosylation sites). This prostate cancer amino acid sequence can be arbitrarily altered by changing the DNA level, in particular by mutation of the DNA encoding the prostate cancer polypeptide at a preselected base, resulting in translation into the desired amino acid. Codons are created.
[0185]
Another means of increasing the number of sugar moieties on the prostate cancer polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods have been described in the art, for example, WO 87/05330, and Aprin and Wriston papers (CRC Crit. Rev. Biochem., 259-306 (1981). )).
[0186]
Removal of the sugar moiety present in a prostate cancer polypeptide can be accomplished by chemical or enzymatic or mutational substitution of codons encoding amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and are described in, for example, Hakimudin et al., Arch. Biochem. Biophys. 259: 52 (1987) and Edge et al., Anal. Biochem. 118: 131 (1981). Enzymatic cleavage of the sugar moiety on the polypeptide is described by Totakara et al., Meth. Enzymol. 138: 350 (1987) and can be achieved by the use of various endoglycosidases and exoglycosidases.
[0187]
Another type of covalent modification of prostate cancer is described in US Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; The prostate cancer polypeptide is linked to one of various non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol or polyoxyalkylene, in a manner as disclosed in 791,192 or 4,179,337. Including that.
[0188]
The prostate cancer polypeptides of the invention can also be modified by methods that form chimeric molecules comprising prostate cancer polypeptides fused to another heterologous polypeptide or amino acid sequence. In certain embodiments, such a chimeric molecule comprises a fusion of a prostate cancer polypeptide with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. This epitope tag is generally located at the amino terminus or carboxyl terminus of the prostate cancer polypeptide. The presence of such epitope-tagged forms of prostate cancer polypeptide can be detected using an antibody against the tag polypeptide. In addition, the supply of epitope tags can allow prostate cancer polypeptides to be readily purified by affinity purification using anti-tag antibodies or by an affinity matrix that binds to another type of epitope tag. In another embodiment, the chimeric molecule may also comprise a fusion of an prostate cancer polypeptide with an immunoglobulin or a specific region of an immunoglobulin. For the bivalent form of the chimeric molecule, such a fusion is the Fc region of an IgG molecule.
[0189]
Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine glycine (poly-his-gly) tags; HIS6 and metal chelate tags, flu HA tag polypeptides and their antibodies 12CA5 (Field et al., Mol. Cell Biol., 8 : 2159-2165 (1988)); c-myc tags and their 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies (Evan et al., Molecular and Cellular Biology, 5: 3610-3616 (1985)); and simple Herpesvirus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering, 3 (6): 547-553 (1990)). Another tag polypeptide is the Flag peptide (Hopp et al., BioTechnology, 6: 1204-1210 (1988)); KT3 epitope peptide (Martin et al., Science, 255: 192-194 (1992)); Tubulin epitope peptide (Skinner J. Biol. Chem., 266: 15163-15166 (1991)); and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87: 6393-6397 (1990). )).
[0190]
Other included are other prostate cancer proteins of the prostate cancer family, and other biological prostate cancer proteins, which are cloned and expressed as outlined below. Thus, probes or degenerate polymerase chain reaction (PCR) primer sequences can be used to find other related prostate cancer proteins from humans or other organisms. As will be appreciated by those skilled in the art, particularly useful probe and / or PCR primer sequences include unique regions of prostate cancer nucleic acid sequences. As is generally known in the art, preferred PCR primers are from about 15 to about 35 nucleotides in length, preferably from about 20 to about 30 and can optionally include inosine. The conditions for the PCR reaction are well known in the art (eg, Innis, PCR protocol, supra).
[0191]
Antibodies against prostate cancer proteins
In a preferred embodiment, when a prostate cancer protein is used for antibody production, such as for immunotherapy or immunodiagnosis, the prostate cancer protein must share at least one epitope or determinant with the full-length protein. . By “epitope” or “determinant” herein is meant a portion of a protein that will typically produce and / or bind to an antibody or T cell receptor in association with MHC. Thus, in most cases, antibodies raised against relatively small prostate cancer proteins will be able to bind to full-length proteins, particularly linear epitopes. In a preferred embodiment, the epitope is unique; that is, antibodies made to the unique epitope show little or no cross-reactivity.
[0192]
Methods for preparing polyclonal antibodies are known to those skilled in the art (eg, Coligan, supra; and Harlow and Lane, supra). Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and / or adjuvant will be injected into the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent can comprise a protein encoded by the illustrated nucleic acids or fragments thereof or fusion proteins thereof. This is useful in conjugating immunizers to proteins that are known to be immunogenic in immunized mammals. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol is selected without undue experimentation by those skilled in the art.
[0193]
Alternatively, the antibody can be a monoclonal antibody. Monoclonal antibodies can be prepared using hybridoma methods, such as those described in Kohler and Milstein, Nature, 256: 495 (1975). In a hybridoma method, a mouse, hamster or other suitable host animal is typically immunized with an immunizer and produces or produces an antibody that specifically binds to the immunizer. Can induce lymphocytes. Alternatively, these lymphocytes are immunized in vitro. This immunizing agent will typically comprise a polypeptide encoded by the nucleic acids of Tables 1-16 and fragments thereof, or a fusion protein thereof. In general, either peripheral blood lymphocytes ("PBL") are used when cells of human origin are desired, or splenocytes or lymph node cells are used when non-human mammalian origin is desired. The lymphocytes are then fused to an immortalized cell line using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, “Monoclonal Antibodies: Principles and Practices”). ", Pp. 59-103 (1986)). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of immortalized cells that are not fused. For example, if the parent cell is deficient in the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for this hybridoma typically contains hypoxanthine, aminopterin and thymidine (“HAT medium”). “), These substances will interfere with the growth of HGPRT-deficient cells.
[0194]
In certain embodiments, the antibody is a bispecific antibody. Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens or that have binding specificities for two epitopes on the same antigen. is there. In certain embodiments, one of the binding specificities relates to a protein encoded by the nucleic acids of Tables 1-16 or fragments thereof, and another relates to any other antigen, and preferably a cell-surface protein or For receptors or receptor subunits, preferably tumor specific. Alternatively, tetramer-type techniques can create multivalent reagents.
[0195]
In a preferred embodiment, an antibody against prostate cancer protein can reduce or eliminate the biological function of prostate cancer protein, as described below. That is, the addition of anti-prostate cancer protein antibodies (either polyclonal or preferably monoclonal) to prostate cancer tissue (or cells containing prostate cancer) can reduce or eliminate prostate cancer. In general, a reduction of at least 25% in activity, growth, size, etc. is preferred, at least about 50% is particularly preferred, and a reduction of about 95-100% is particularly preferred.
[0196]
In a preferred embodiment, the antibody to prostate cancer protein is a humanized antibody (eg, Xenelex Biosciences, Mederex, Abgenix, Protein Design Labs). Humanized forms of non-human (eg murine) antibodies are immunoglobulins, immunoglobulin chains or fragments thereof (eg Fv, Fab, Fab ′, F (ab ′) 2 or It is a chimeric molecule of an antigen-binding partial sequence of another antibody. Humanized antibodies are derived from non-human species (donor antibodies) such as mice, rats or rabbits in which residues from the complementarity determining regions (CDRs) of the recipient have the desired specificity, affinity and capacity. Includes human immunoglobulins (recipient antibodies), such as those exchanged for residues from the CDRs. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody comprises substantially all of at least one, and typically two variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin. And all or substantially all framework (FR) regions are of human immunoglobulin consensus sequence. Humanized antibodies will most suitably contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321: 522-525 (1986). Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992)). Humanization can be performed essentially according to the method of Winter and colleagues, replacing the rodent CDR or CDR sequence with the sequence of the corresponding human antibody (Jones et al., Nature, 321: 522-525 ( 1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)). Accordingly, such humanized antibodies are chimeric antibodies (US Pat. No. 4,816,567), wherein substantially less than fully human variable domains are replaced with sequences from the corresponding non-human species. The
[0197]
Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991)). The techniques of Cole et al. And Boerner et al. Are also available for the preparation of human monoclonal antibodies (Cole et al., “Monoclonal Antibodies and Cancer Therapy”, page 77 and Boerner et al., J. Immunol. 147 ( 1): 86-95 (1991)). Similarly, human antibodies can be generated by introduction of human immunoglobulin loci into transgenic animals, such as mice in which endogenous immunoglobulin genes are partially or completely inactivated. Upon challenge, human antibody production is observed, which is very similar to that observed in humans in all respects, including gene rearrangement, assembly and antibody repertoire. This method is described, for example, in US Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 661,016, and the following scientific publications: Marks et al., Bio / Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859 (1994); Morrison ( Morrison, Nature, 368: 812-13 (1994); Fishwild et al., Nature Biotechnology, 14: 845-51 (1996); Neuberger, Nature, Biotechnology, 14: 826 (1996); Ron Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).
[0198]
Immunotherapy refers to the treatment of prostate cancer with antibodies raised against prostate cancer protein. The immunotherapy used herein can be passive or active. As defined herein, passive immunotherapy passively transfers antibodies to a recipient (patient). Active immunization is the induction of antibody and / or T cell responses in a recipient (patient). Induction of the immune response is the result of providing a recipient with the antigen against which the antibody was raised. As will be appreciated by those skilled in the art, this antigen may be an antigen expression of a nucleic acid capable of expressing the antigen with the recipient, or injection of a polypeptide against which it is desired that antibodies be raised in the recipient. Can be provided by contact under conditions for leading to an immune response.
[0199]
In a preferred embodiment, the prostate cancer protein against which antibodies are raised is a secreted protein as described above. Although there is no theoretical support, the antibody used for therapy binds to the secreted protein and prevents it from binding to the receptor, thereby inactivating the secreted prostate cancer protein.
[0200]
In another preferred embodiment, the prostate cancer protein against which antibodies are raised is a transmembrane protein. Although not theoretically supported, antibodies used in therapy bind to the extracellular domain of prostate cancer protein, preventing it from binding to other proteins such as circulating ligands or cells-related molecules. This antibody can cause downregulation of the transmembrane prostate cancer protein. As will be appreciated by those skilled in the art, this antibody is a competitive, non-competitive or uncompetitive inhibitor of a protein that binds to the extracellular domain of prostate cancer protein. This antibody is also an antagonist of prostate cancer protein. Furthermore, this antibody prevents activation of the transmembrane prostate cancer protein. In certain aspects, the antibody interferes with cell growth if the antibody interferes with the binding of other molecules to the prostate cancer protein. This antibody may be a cytotoxic agent, including but not limited to TNF-α, TNF-β, IL-1, INF-γ and IL-2, or 5FU, vinblastine, actinomycin D, cisplatin It can also be used to target or sensitize cells to chemotherapeutic agents such as methotrexate. In some cases, the antibody belongs to a subtype that activates serum complement when complexed with a transmembrane protein, thereby mediating cytotoxicity or antigen-dependent cytotoxicity (ADCC). Accordingly, prostate cancer is treated by administering to the patient an antibody against a transmembrane prostate cancer protein. Antibody labeling provides a means to activate co-toxin, localize the toxin payload, or otherwise remove cells locally.
[0201]
In another preferred embodiment, the antibody is conjugated to an effector moiety. The effector moiety can be a number of molecules, including a labeling site such as a radiolabel or a fluorescent label, and can be a therapeutic moiety. In certain aspects, the therapeutic moiety is a small molecule that modulates the activity of prostate cancer protein. In another aspect, the therapeutic moiety modulates the activity of molecules associated with or in close proximity to prostate cancer proteins. This therapeutic moiety can inhibit enzyme activity such as protease or collagenase or protein kinase activity associated with prostate cancer.
[0202]
In preferred embodiments, the therapeutic moiety can also be a cytotoxic agent. In this method, targeting of cytotoxic agents to prostate cancer tissue or cells reduces the number of affected cells, thereby reducing symptoms associated with prostate cancer. Cytotoxic agents are numerous, variable, and include, but are not limited to, cytotoxic drugs or toxins or active fragments of such toxins. Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin and the like. Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antibodies raised against prostate cancer proteins, or by conjugation with a chelating agent covalently bound to the radionuclide antibody. Targeting a therapeutic moiety to a transmembrane prostate cancer protein not only increases the local concentration of the therapeutic moiety in the area affected by prostate cancer, but also reduces adverse side effects that may be associated with the therapeutic moiety Also useful.
[0203]
In another preferred embodiment, the prostate cancer protein against which antibodies are raised is an intracellular protein. In this case, the antibody may be conjugated to a protein that promotes entry into the cell. In some cases, the antibody enters the cell by endocytosis. In another embodiment, a nucleic acid encoding the antibody is administered to the individual or cell. Furthermore, if the prostate cancer protein is targeted intracellularly, i.e. in the nucleus, the antibody additionally comprises a target localization signal, i.e. a nuclear localization signal.
[0204]
The prostate cancer antibody of the present invention specifically binds to prostate cancer protein. As used herein, “specific binding” refers to K_dMeans that the antibody binds to the protein at least about 0.1 mM, more typically at least about 1 μM, preferably at least about 0.1 μM or more, or most preferably 0.01 μM or more. Bond selectivity is also important.
[0205]
Detection of prostate cancer sequences for diagnostic and therapeutic applications
In certain aspects, the RNA expression level of the gene is determined for different cellular states of the prostate cancer phenotype. Expression levels of genes are evaluated and expressed in normal tissue (ie, not prostate cancer) and prostate cancer tissue (and possibly for the varying severity of prostate cancer associated with prognosis as outlined below) Provide a profile. The expression profile of a particular cell state or point of development is essentially a “fingerprint” of that state. While the two states have specific genes expressed as well, the evaluation of many genes allows the creation of gene expression profiles that simultaneously reflect the state of the cells. By comparing the expression profiles of cells in different states, information about which genes are important in each of these states (including both up- and down-regulation of genes) is obtained. A determination is then made whether a diagnosis is made or whether the tissue sample has a gene expression profile of normal or cancerous tissue. This will provide molecular diagnosis of the relevant condition.
[0206]
As used herein, “differential expression” or grammatical equivalent means a qualitative or quantitative difference in temporal and / or cellular gene expression patterns within and between cells and tissues. . Thus, a differentially expressed gene can have its expression altered qualitatively, including activation or deactivation, for example, in normal versus prostate cancer tissue. A gene may be activated or stopped in a particular state compared to another state, thus allowing a comparison of two or more states. A qualitatively regulated gene will exhibit an expression pattern in a state or cell type detectable by standard techniques. Some genes are expressed in one state or cell type, but not in both. Alternatively, the difference in expression is quantitative, for example, in that expression increases or decreases; that is, gene expression is up-regulated, resulting in increased amounts of transcript or down-regulated, decreased amounts of transcript. Produce. The degree of expression differs to the standard described in detail below, such as the Affymetrix GeneChip ™ expression array (Lockhart, Nature Biotechnology, 14: 1675-1680 (1996), which is expressly incorporated by reference). It is only necessary to be large enough to be quantified by the characterization technique. Other techniques include, but are not limited to, quantitative reverse transcriptase PCR, Northern analysis, and RNase protection. As outlined above, a preferred change in expression (ie, upregulation or downregulation) is at least about 50%, more preferably at least about 100%, more preferably at least about 150%, more preferably at least about 200%. With 300 to at least 1000% being particularly preferred.
[0207]
Evaluation can be done on the gene transcript or protein level. The amount of gene expression can be monitored using nucleic acid probes to the DNA or RNA equivalent of the gene transcript, or quantification of gene expression levels, or the final gene product itself (protein), eg, prostate It can be monitored by antibodies to cancer proteins and other techniques including standard immunoassay methods (such as ELISA) or mass spectrometry assays, 2D gel electrophoresis assays, and the like. Proteins corresponding to prostate cancer genes, ie those identified as important in the prostate cancer phenotype, can be evaluated in prostate cancer diagnostic tests.
[0208]
In a preferred embodiment, gene expression monitoring is performed on many genes simultaneously. In addition, multiple protein expression can be monitored. Similarly, these assays can be performed on an individual basis.
[0209]
In this embodiment, a prostate cancer nucleic acid probe is attached to the biochip outlined herein for detection and quantification of prostate cancer sequences in specific cells. This assay is further described in the examples below. PCR techniques can be used to provide better sensitivity.
[0210]
In a preferred embodiment, a nucleic acid encoding prostate cancer protein is detected. Although DNA or RNA encoding prostate cancer protein can be detected, of particular interest is the method by which mRNA encoding prostate cancer protein is detected. Probes that detect mRNA are nucleotide / deoxynucleotide probes that are complementary to and hybridize to mRNA, and include, but are not limited to, oligonucleotides, cDNA or RNA. The probe may also include a detectable label, as defined herein. In some methods, mRNA is detected after the test nucleic acid is immobilized on a solid support, such as a nylon membrane, and the probe is hybridized to the sample. After washing away non-specifically bound probe, this label is detected. In the alternative, the detection of mRNA is performed in situ. In this method, the permeabilized cell or tissue sample is contacted with a detectable labeled nucleic acid probe for a time sufficient for the probe to hybridize to the target mRNA. After washing away non-specifically bound probe, the label is detected. For example, a digoxigenin-labeled riboprobe (RNA probe) that is complementary to mRNA encoding prostate cancer protein binds digoxigenin to an anti-digoxigenin secondary antibody, as well as nitroblue tetrazolium and 5-bromo-4- Detected by coloration with chloro-3-indoyl phosphate.
[0211]
In a preferred embodiment, various proteins from the three classes of proteins described herein (secreted, transmembrane or intracellular proteins) can be used in diagnostic assays. Cells containing prostate cancer proteins, antibodies, nucleic acids, modified proteins and prostate cancer sequences are used in diagnostic assays. This can be done at the individual gene or corresponding polypeptide level. In preferred embodiments, expression profiles are used, preferably in combination with high throughput screening techniques that allow for monitoring of expression profile genes and / or corresponding polypeptides.
[0212]
As described and defined herein, prostate cancer proteins, including intracellular, transmembrane or secreted proteins, have found use as markers for prostate cancer. Detection of these proteins in tissues expected to be prostate cancer allows detection or diagnosis of prostate cancer. In certain embodiments, antibodies are used to detect prostate cancer protein. A preferred method is to separate the protein from the sample by electrophoresis on a gel (typically a denatured and reduced protein gel, but can be other gel types including isoelectric gels, etc.). After separation of the protein, the prostate cancer protein is detected, for example, by immunoblotting with an antibody against the prostate cancer protein. Immunoblotting methods are well known to those skilled in the art.
[0213]
In another preferred method, antibodies against prostate cancer proteins find use in, for example, in situ imaging techniques in histology (eg, “Methods in Cell Biology: Antibodies in Cell Biology: Antibodies in Cell Biology”). 37] (Edited by Asai, 1993)). In this method, the cells are contacted with one to several antibodies against prostate cancer protein. After washing away non-specific antibody binding, the presence of one or more antibodies is detected. In certain embodiments, the antibody is detected by incubating with a secondary antibody that includes a detectable label. Alternatively, the primary antibody against prostate cancer protein comprises a detectable label, eg, an enzyme marker that can act on the substrate. In another preferred embodiment, each of the plurality of primary antibodies comprises a distinguished and detectable label. This method finds use in the simultaneous screening of multiple prostate cancer proteins. Many other histological imaging techniques are also provided by the present invention, as will be appreciated by those skilled in the art.
[0214]
In a preferred embodiment, the label is detected by a fluorometer that has the ability to detect and discriminate the emission of various wavelengths. In addition, a fluorescence activated cell sorter (FACS) can be used in the method.
[0215]
In another preferred embodiment, the antibody finds use in the diagnosis of prostate cancer from blood, serum, plasma, stool, and other samples. Such samples are therefore useful as samples to be probed or tested for the presence of prostate cancer protein. The antibodies can be used for detection of prostate cancer proteins by previously described immunoassay techniques including ELISA, immunoblotting (Western blotting), immunoprecipitation, BIACORE techniques and the like. In contrast, the presence of antibodies may indicate an immune response against endogenous prostate cancer protein.
[0216]
In a preferred embodiment, in situ hybridization is performed to a tissue array of labeled prostate cancer nucleic acid probes. For example, a tissue sample array is created that includes prostate cancer tissue and / or normal tissue. In situ hybridization (eg, Ausubel, supra) is then performed. In comparing fingerprints between individuals and standards, one skilled in the art can make a diagnosis, prognosis, or prediction based on the findings. In addition, the genes that indicate this diagnosis may differ from those that indicate prognosis, and molecular profiling of the cellular state distinguishes between treatment responsive or resistant states or predicts outcome It is understood that it can be done.
[0217]
In preferred embodiments, cells comprising prostate cancer proteins, antibodies, nucleic acids, modified proteins and prostate cancer sequences are used in prognostic assays. As described above, gene expression profiles correlated with prostate cancer can be generated for long-term prognosis determination. Again this can be done either at the protein or gene level, the use of genes being preferred. As described above, a prostate cancer probe can be attached to a biochip for detection and quantification of prostate cancer sequences in a tissue or patient. This assay proceeds as outlined above for diagnosis. PCR methods can provide more sensitive and accurate quantification.
[0218]
Therapeutic Compound Assays
In preferred embodiments, members of the proteins, nucleic acids and antibodies described herein are used in drug screening assays. Cells containing prostate cancer proteins, antibodies, nucleic acids, modified proteins and prostate cancer sequences are used in drug screening assays or by assessing the effect of drug candidates on “gene expression profiles” or polypeptide expression profiles. The In preferred embodiments, expression profiles are used, preferably in combination with high-throughput screening techniques that allow monitoring of expression profile genes after treatment with candidate substances (eg, Zlokarnik et al., Science, 279: 84- 8 (1998); Heid, Genome Res., 6: 986-94 (1996)).
[0219]
In preferred embodiments, cells comprising prostate cancer proteins, antibodies, nucleic acids, modified proteins and native or modified prostate cancer proteins are used in screening assays. That is, the present invention provides a novel screening method for the physiological function of a composition that modulates the prostate cancer phenotype or the identified prostate cancer protein. As mentioned above, this can be done at the individual gene level or by assessing the effects of drug candidates on a “gene expression profile”. In a preferred embodiment, an expression profile is used, preferably in combination with a high-throughput screening technique that allows monitoring of expression profile genes after treatment with a candidate substance, which is described in Zlokarnik et al. )checking.
[0220]
Genes that are differentially expressed herein can be identified and various assays can be performed. In preferred embodiments, the assay can be attempted at the individual gene or protein level. That is, once a particular gene is identified as an upregulation in prostate cancer, test compounds can be screened for the ability to modulate gene expression or bind to prostate cancer protein. “Modulation” thus includes both an increase and a decrease in gene expression. The preferred amount of modulation depends on the initial change in gene expression in normal versus prostate cancer tissue, this change being at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300. It appears to be ~ 1000% or more. Thus, if the gene shows a 4-fold increase in prostate cancer tissue compared to normal tissue, a reduction of approximately 4-fold is often desirable; similarly, a 10-fold decrease in prostate cancer tissue compared to normal tissue Often provides a target value of a 10-fold increase in expression to be induced by the test compound.
[0221]
The amount of gene expression is monitored using a nucleic acid probe, and the level of gene expression, or the gene product itself, is monitored, for example, by use of antibodies against prostate cancer proteins and standard immunoassays. Proteomics and separation techniques also allow quantification of expression.
[0222]
In a preferred embodiment, the gene expression or protein monitoring of many entities, ie the expression profile, is monitored simultaneously. Such a profile would typically be associated with a plurality of those entities described herein.
[0223]
In this embodiment, a prostate cancer nucleic acid probe is attached to the biochip as outlined below for detection and quantification of prostate cancer sequences in specific cells. Alternatively, PCR can be used. Thus, for example, a series of microtiter plates can be used with primers distributed in the desired wells. A PCR reaction is then performed and analyzed for each well.
[0224]
Expression monitoring can be performed to identify compounds that modify the expression of sequences associated with one or more prostate cancers, such as the polynucleotide sequences shown in Tables 1-16. In general, in a preferred embodiment, a test modulator is added to the cells prior to analysis. Screening to identify substances that further modulate prostate cancer, substances that modulate prostate cancer proteins, substances that bind to prostate cancer proteins, or substances that interfere with the binding of prostate cancer proteins and antibodies or other binding partners Is also provided.
[0225]
As used herein, the term “test compound” or “drug candidate” or “modulator” or grammatical equivalents directly express prostate cancer phenotype or prostate cancer sequence expression, eg, nucleic acid or protein sequence expression. Any molecule to be tested for the ability to alter, either indirectly or indirectly, such as proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, etc. is described. In preferred embodiments, the modulator alters the expression profile or the nucleic acid or protein of the expression profile provided herein. In certain embodiments, the modulator suppresses a prostate cancer phenotype, eg, for a normal tissue fingerprint. In another embodiment, the modulator induces a prostate cancer phenotype. In general, multiple assay mixtures are tried in parallel at different substance concentrations to obtain differential responses to various concentrations. Typically, one of these concentrations is utilized as a negative control, ie zero concentration or below the detection level.
[0226]
Drug candidates encompass many chemical classes, but typically these are organic molecules, preferably small organic compounds with a molecular weight of greater than 100 daltons and less than about 2,500 daltons. Preferred small molecules are less than 2000D, or less than 1500D, or less than 1000D or less than 500D. Candidate substances contain functional groups necessary for structural interactions with proteins, especially hydrogen bonds, typically contain at least amine, carbonyl, hydroxyl or carboxyl groups, preferably contain at least two functional chemical groups . This candidate substance often contains a cyclic carbon or heterocyclic structure and / or an aromatic or polyaromatic structure substituted with one or more of the above functional groups. Candidate substances are also found in biomolecules including peptides, carbohydrates, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.
[0227]
In certain aspects, the modulator appears to neutralize the action of prostate cancer proteins. “Neutralization” means that the activity of the protein is inhibited or blocked, resulting in an effect on the cell.
[0228]
In certain embodiments, combinatorial libraries of potential modulators are screened for the ability to bind to prostate cancer polypeptides or to modulate activity. Typically, a new chemical entity with useful properties identifies a chemical compound (referred to as a “lead compound”) for some desirable property or activity, eg, inhibitory activity, produces a variant of the lead compound, and It is created by evaluating the properties and activities of these variant compounds. Often, high throughput screening (HTS) methods are utilized for such analysis.
[0229]
In certain preferred embodiments, the high-throughput screening method provides a library containing a large number of potential therapeutic compounds (candidate compounds). Such “combinatorial chemical libraries” are then screened by one or more assays that identify library members (specific chemical species or subclasses) that exhibit the desired characteristic activity. The compounds thus identified can be utilized as normal “lead compounds” or can themselves be used in promising or actual therapy.
[0230]
A combinatorial chemical library is a collection of diverse chemical compounds created by either chemical synthesis or biological synthesis, with a combination of many chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide (eg, mutein) library, can be used for each possible method for a given compound length (ie, the number of amino acids in a polypeptide compound). Created by a combination of a set of chemical building blocks, called amino acids. Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks (Gallop et al., J. Med Chem., 37 (9): 1233-1251 (1994)).
[0231]
The preparation and screening of combinatorial chemical libraries is well known to those skilled in the art. Such combinatorial chemical libraries are peptide libraries (eg, US Pat. No. 5,010,175, Furka, Pept. Prot. Res., 37: 487-493 (1991), Houghton et al., Nature, 354: 84-88 (1991)), peptoids (PCT International Publication No. 91/19735), encoded peptides (PCT International Publication No. 93/20242), random biooligomers (PCT International Publication No. 92/00091). ), Benzodiazepines (US Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA, 90: 6909-6). 13 (1993)), vinyl polypeptides (Hagihara et al., J. Amer. Chem. Soc., 114: 6568 (1992)), non-peptidic peptide mimetics with β-D-glucose scaffolds (Hirschmann et al., J Amer. Chem. Soc., 114: 9217-9218 (1992)), analog organic synthesis of small compound libraries (Chen et al., J. Amer. Chem. Soc., 116: 2661 (1994)), oligocarbamates (Cho et al., Science, 261: 1303 (1993)), and / or peptidyl phosphate (Campbell et al., J. Org. Chem., 59: 658 (1994)). . In general, Gordon et al. (J. Med. Chem., 37: 1385 (1994)), nucleic acid libraries (see, eg, Stratgene), peptide nucleic acid libraries (eg, US Pat. No. 5,539,083). (See, eg, Vaughn et al., Nature Biotechnology, 14 (3): 309-314 (1996), and PCT / US96 / 10287), carbohydrate libraries (eg, Liang et al., Science, 274: 1520). -1522 (1996), and US Pat. No. 5,593,853), and small organic molecule libraries (eg, benzodiazepine, Baum, C & EN, Jan. 18, p. 33 (1993); isoprenoids, US Pat. 5,569, 588; thiazolidinones and metathiazonones, US Pat. No. 5,549,974; pyrrolidines, US Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, US Pat. No. 5,506,337; See beazodiazepine, US Pat. No. 5,288,514;
[0232]
Equipment for combinatorial library preparation is commercially available (eg, 357 MPS, 390 MPS, Advanced Chem Tech, Lewisville (KY), Symphony, Rainin, Woburn (MA), 433A, Applied Biosystems, (See Foster City (CA), 9050 Plus, Millipore, Bedford (MA)).
[0233]
Many well-known robotic systems have also been developed for liquid phase chemistry. These systems include automated workstations, such as automated synthesizers developed by Takeda Chemical Industries (Osaka, Japan), as well as many robotic systems that use robotic arms (Zymate II, Zymark). Corp., Hopkinton, MA; Orca, Hewlett-Packard, Palo Alto, CA), which mimics a manual synthesis operation performed by chemists. Any of the above devices are suitable for use with the present invention. The nature and performance (if any) of the improvement of these devices to be able to operate as described herein will be apparent to those skilled in the relevant art. In addition, many combinatorial libraries are commercially available themselves (eg, ComGenex, Princeton (NJ), Asinex, Moscow (Russia), Tripos, St. Louis (MO), ChemStar, Moscow (Russia), 3D Pharmaceuticals, Exton (PA), Martek Biosciences, Colombia (MD), etc.)
[0234]
Assays that identify modulators are amenable to high-throughput screening. Accordingly, preferred assay methods detect enhancement or inhibition of prostate cancer gene transcription, inhibition or enhancement of polypeptide expression, and inhibition or enhancement of polypeptide activity.
[0235]
High throughput assays for the presence, absence, quantification, or other properties of a particular nucleic acid or protein product are well known to those skilled in the art. Similarly, binding assays and reporter gene assays are similarly well known. Thus, for example, US Pat. No. 5,559,410 discloses a high-throughput screening method for proteins and US Pat. No. 5,585,639 discloses a high-throughput screening method for nucleic acid binding (ie, in an array). Meanwhile, US Pat. Nos. 5,576,220 and 5,541,061 disclose high-throughput methods for screening for ligand / antibody binding.
[0236]
In addition, high-throughput screening systems are commercially available (see, for example, Zymark, Hopkinton, MA; Air Technical Industries, Menthol, OH; Beckman Instruments, Frelton, CA; Precision Systems, Natick, MA, etc.). These systems are typically automated at all stages, including all pipetting of samples and reagents, liquid dispensing, incubation for a period of time, and final reading of the microplate in a detector suitable for the assay. Yes. These configurable systems provide high throughput and rapid start-up, as well as a high degree of flexibility and custom manufacturing. The manufacturers of such systems provide detailed protocols for various high throughput systems. Thus, for example, Zymark provides a technical report describing screening systems that detect modulation of gene transcription, ligand binding, and the like.
[0237]
In certain embodiments, the modulator is a protein, many of which are natural proteins or fragments of natural proteins. Thus, for example, cell extracts containing proteins, or random or designated digests of proteinaceous cell extracts can be used. In this method, a protein library for screening with the method of the present invention is created. Particularly preferred in this embodiment are bacterial, fungal, viral, and mammalian protein libraries, the latter being preferred and human proteins being particularly preferred. Particularly useful test compounds will be related to the type of protein to which the target belongs, such as enzyme or ligand substrates and receptors.
[0238]
In a preferred embodiment, the modulator is a peptide of about 5 to about 30 amino acids, with about 5 to about 20 amino acids being preferred, and about 7 to about 15 being particularly preferred. These peptides may be digests of natural proteins as described above, random peptides, or “biased” random peptides. As used herein, “randomized” or grammatical equivalents mean that each nucleic acid and peptide consists essentially of random nucleotides and amino acids, respectively. Generally, these random peptides (or nucleic acids discussed below) are chemically synthesized so that they are incorporated into any nucleotide or amino acid at any position. This synthesis process can be designed to create randomized proteins or nucleic acids to allow the formation of all or most possible combinations beyond the length of the sequence, and as a result randomized. A library of candidate bioactive proteinaceous materials is formed.
[0239]
In certain embodiments, the library is completely randomized and there is no sequence preference or constant at any position. In a preferred embodiment, the library is biased. That is, some positions in the array are either kept constant or selected from the possibility of the current constant. For example, in a preferred embodiment, the nucleotide or amino acid residue is directed towards, for example, a hydrophobic amino acid, a hydrophilic residue, a sterically biased (either small or large) residue, certain nucleic acid binding domains, Randomized to limited types such as cysteine formation, cross-linking, proline for SH-3 domain, serine, threonine, tyrosine or histidine for phosphorylation sites, or purines.
[0240]
The modulator of prostate cancer may be a nucleic acid as defined below. In general, as indicated above for proteins, the nucleic acid modulator can be a natural nucleic acid, a random nucleic acid, or a “biased” random nucleic acid. For example, prokaryotic or eukaryotic genome digestion can be used as outlined above for proteins.
[0241]
In certain embodiments, the activity of a protein associated with prostate cancer is an antisense polynucleotide, i.e., a nucleic acid complementary to an encoding mRNA nucleic acid sequence or a subsequence thereof, such as prostate cancer protein mRNA, and to these. By the use of nucleic acids that preferentially specifically hybridize, it is down-regulated or totally inhibited. Binding of the antisense polynucleotide to the mRNA reduces the translation and / or stability of the mRNA.
[0242]
In the context of the present invention, antisense polynucleotides can include natural nucleotides, or synthetic species formed from natural subunits or their close homologues. Antisense polynucleotides can also have altered sugar moieties or internal sugar linkages. Among these are phosphorothioates and other sulfur-containing species that are known for use in the art. Analogs are encompassed by the present invention as long as they function effectively to hybridize to prostate cancer protein mRNA. See, for example, Isis Pharmaceuticals, Carlspad, CA; Sequiter, Natick, MA.
[0243]
Such antisense polynucleotides can be readily synthesized using recombinant means or synthesized in vitro. Such synthesizers are sold by several specialist vendors, including Applied Biosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those skilled in the art.
[0244]
Antisense molecules as used herein include antisense or sense oligonucleotides. Sense oligonucleotides can be used, for example, to block transcription by binding to the antisense strand. Antisense and sense oligonucleotides comprise a single stranded nucleic acid sequence (either RNA or DNA) that is capable of binding to a target mRNA (sense) or DNA (antisense) sequence of a prostate cancer molecule. In accordance with the present invention, an antisense or sense oligonucleotide comprises a fragment, generally at least about 14 nucleotides, preferably about 14-30 nucleotides. The ability to derive antisense or sense oligonucleotides based on a cDNA sequence encoding a given protein is described, for example, by Stein and Cohen (Cancer Res., 48: 2659 (1988)). And van der Krol et al. (BioTechniques, 6: 958 (1988)).
[0245]
In addition to antisense polynucleotides, ribozymes can be used to target nucleotide sequences associated with prostate cancer and inhibit transcription. Ribozymes are RNA molecules that catalytically cleave other RNA molecules. Various types of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNase P, and axhead ribozymes (for general verification of the properties of various ribozymes, for example Castanototo et al., Adv., Pharmacology, 25: 289-317 (1994)).
[0246]
General features of hairpin ribozymes are described, for example, in the paper by Hampel et al. (Nucl. Acids Res., 18: 299-304 (1990)); European Patent Publication No. 0 360 257; US Pat. No. 5,254. , 678. Methods of preparation are well known to those skilled in the art (eg, WO 94/26877; Ojwang et al., Proc. Natl. Acad Sci. USA, 90: 6340-6344 (1993); Yamada et al., Human Gene Therapy, 1: 39-45 (1994); Leavitt et al., Proc. Natl Acad Sci. USA, 92: 699-703 (1995); Leavitt et al., Human Gene Therapy, 5: 1151-120 (1994); and Yamada et al., Virology, 205: 121-126 (1994)).
[0247]
Prostate cancer polynucleotide modulators can be introduced into cells containing the target nucleotide sequence by formation of a complex with a ligand binding molecule as disclosed in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, the ligand binding molecule complex does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or is a sense or antisense oligonucleotide or complex thereof. Does not block entry into cells. Alternatively, prostate cancer polynucleotide modulators can be introduced into cells containing the target nucleic acid sequence by formation of a polynucleotide-lipid complex, for example, as disclosed in WO 90/10448. It will be appreciated that the use of antisense molecules or knock-out and knock-in models can also be used in screening assays as described above in addition to therapeutic methods.
[0248]
As noted above, gene expression monitoring is conveniently used for testing candidate modulators (eg, proteins, nucleic acids or small molecules). After the candidate substance is added and the cells are incubated for a period of time, a sample containing the target sequence to be analyzed is added to the biochip. If necessary, this target sequence is prepared using known techniques. For example, this sample can be treated to lyse cells using a known lysis buffer, electroporation or the like by appropriate purification and / or amplification such as PCR. For example, in vitro transcription with a label covalently attached to a nucleotide is performed. In general, nucleic acids are labeled with biotin FITC or PE, or cy3 or cy5.
[0249]
In preferred embodiments, the target sequence is labeled, eg, with a fluorescent, chemiluminescent, chemical, or radioactive signal, to provide a means of detecting specific binding of the target sequence to the probe. The label can also be an enzyme, such as alkaline phosphatase or horseradish peroxidase, which, when provided with a suitable substrate, produces a product that can be detected. Alternatively, the label can be a labeled compound or small molecule, such as an enzyme inhibitor, that binds to the enzyme but does not catalyze or alter it. The label can also be a moiety or compound such as biotin that specifically binds to an epitope tag or streptavidin. For the biotin example, streptavidin is labeled as described above, which provides a detectable signal for the bound target sequence. Unbound labeled streptavidin is typically removed prior to analysis.
[0250]
As will be appreciated by those skilled in the art, these assays may be direct hybridization assays or may include “sandwich assays” involving the use of multiple probes, which are generally described in US Pat. , 681,702, 5,597,909, 5,545,730, 5,594,117, 5,591,584, 5,571,670, 5,580 No. 7,731, No. 5,571,670, No. 5,591,584, No. 5,624,802, No. 5,635,352, No. 5,594,118, No. 5,359,100 No. 5,124,246 and 5,681,697, all of which are incorporated herein by reference. In this embodiment, generally, the target nucleic acid is prepared as described above and then added to the biochip containing the plurality of nucleic acid probes under conditions that can form a hybridization complex.
[0251]
A variety of hybridization conditions can be used in the present invention, including high, medium and low stringency conditions as outlined above. These assays are generally performed under stringency conditions such that a labeled probe hybridization complex is formed only when the target is present. Stringency can be achieved by changing the stage parameters, which are thermodynamic variables, including but not limited to temperature, formamide concentration, salt concentration, chaotropic salt concentration, pH, organic solvent concentration, etc. Can be controlled.
[0252]
These parameters can also be used for control of non-specific binding, as more generally disclosed in US Pat. No. 5,681,697. Therefore, it is desirable to perform the steps at relatively high stringency conditions that reduce non-specific binding.
[0253]
The reactions outlined herein can be realized in a variety of ways. The components of this reaction can be added simultaneously or sequentially in a different order according to the preferred embodiments outlined below. In addition, the reaction can include a variety of other reagents. These include salts, buffers, neutral proteins such as albumin, detergents, etc., to facilitate optimal hybridization and detection, and / or reduce non-specific or background interactions. used. Otherwise, reagents that improve assay efficiency, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like, can be used as appropriate depending on the sample preparation method and target purity.
[0254]
This assay data is analyzed to determine the expression level of individual genes, changes in expression levels between states, and form gene expression profiles.
[0255]
Screening is done to identify modulators of the prostate cancer phenotype. In one embodiment, the screening is performed to identify modulators that can induce or suppress a particular expression profile, and thus preferably create an associated phenotype. In other embodiments, such as diagnostic applications, differentially expressed genes that are important in a particular condition are identified, and screening can be performed to identify modulators that alter the expression of individual genes. . In another embodiment, the screening is performed to identify modulators that alter the biological function of the expression product of the differentially expressed gene. In addition, the importance of the gene in a particular state is identified, and screening is performed to identify substances that bind to the genetic product and / or modulate biological activity.
[0256]
In addition, screening can be performed for genes that induce a response to a candidate substance. After identifying a modulator based on its ability to suppress the prostate cancer expression pattern leading to a normal expression pattern, or to modulate a single prostate cancer gene expression profile to mimic the expression of a gene from normal tissue, Screening can be performed to identify genes that are specifically modulated in response to this agent. Comparison of expression profiles between normal tissue and prostate cancer tissue treated with material reveals genes that are not expressed in normal tissue or prostate cancer tissue but are expressed in tissue treated with material. These substance-specific sequences are identified and used by the methods described herein for prostate cancer genes or proteins. In particular, these sequences and the proteins they encode find use in the marking or identification of cells treated with substances. In addition, antibodies can be used to target new therapies against prostate cancer tissue samples raised against and treated with this substance-derived protein.
[0257]
Thus, in certain embodiments, a test compound is administered to a population of prostate cancer cells that has an associated prostate cancer expression profile. As used herein, “administration” or “contact” is a method that allows a candidate substance to act on a cell either by uptake and intracellular action or by action at the cell surface. Is added to the cells. In some embodiments, a nucleic acid encoding a proteinaceous candidate substance (ie, a peptide) is placed in a viral construct, such as an adenovirus or retroviral construct, for example as described in PCT / US97 / 01019. And added to the cell, so that expression of this peptide substance is realized. An adjustable gene therapy system can also be used.
[0258]
Once the test compound is administered to the cells, the cells can be washed if desired and incubated for a period of time, preferably under physiological conditions. These cells are then collected and a new gene expression profile is created as outlined herein.
[0259]
Thus, for example, prostate cancer tissue is screened for substances that modulate, eg, induce or suppress, the prostate cancer phenotype. Changes in at least one of the expression profiles, preferably many genes, indicate that this substance has an effect on prostate cancer activity. By defining such prostate cancer phenotype signatures, screening for new drugs that alter the phenotype can be devised. In this method, the drug target need not be known and need not be presented in the original expression screening platform, nor does the target protein transcript level need to change.
[0260]
In preferred embodiments, as outlined above, screening can be performed on individual genes and gene products (proteins). That is, a specific differentially expressed gene is identified as important in a particular state, and a modulator of its own expression of either the gene or gene product can be screened. The gene product of a differentially expressed gene is sometimes referred to herein as “prostate cancer protein” or “prostate cancer modulating protein”. The prostate cancer modulating protein can be a fragment encoded by the nucleic acids of Tables 1-16, or it can be a full-length protein for the fragment. Preferably, the prostate cancer modulating protein is a fragment. In preferred embodiments, the prostate cancer amino acid sequences used to determine sequence identity or similarity are encoded by the nucleic acids of Tables 1-16. In another embodiment, these sequences are natural allelic variants of the proteins encoded by the nucleic acids of Tables 1-16. In another embodiment, these sequences are sequence variants as further described herein.
[0261]
Preferably, the prostate cancer modulating protein is a fragment of approximately 14-24 amino acids in length. More preferably, this fragment is a soluble fragment. Preferably, this fragment comprises a non-transmembrane region. In a preferred embodiment, this fragment has an N-terminal Cys to aid solubility. In some embodiments, the C-terminus of this fragment is maintained as a free acid and the N-terminus is a free amine to aid coupling, ie cysteine.
[0262]
In certain embodiments, the prostate cancer protein is conjugated to an immunogenic agent as discussed herein. In certain embodiments, the prostate cancer protein is conjugated to BSA.
[0263]
Measurement of prostate cancer polypeptide activity, or prostate cancer or prostate cancer phenotype can be performed using a variety of assays. For example, the effect of a test compound on prostate cancer polypeptide function can be measured by testing the parameters as described above. Appropriate physiological changes that affect activity can be used to assess the effect of test compounds on the polypeptides of the invention. Tumor, tumor growth, tumor metastasis, angiogenesis, hormone release, transcriptional changes to known but uncharacterized genetic markers (eg, Northern blots) when functional outcome is determined using intact cells or animals ), Changes in cellular metabolism, such as changes in cell growth or pH, as well as various effects in prostate cancer cases associated with changes in intracellular second messengers such as cGMP. In the assay method of the present invention, a mammalian prostate cancer polypeptide is typically used, for example a mouse, preferably a human.
[0264]
Assays to identify compounds that modulate activity can be performed in vitro. For example, a prostate cancer polypeptide is first contacted with a potential modulator and incubated for an appropriate time, such as 0.5 to 48 hours. In certain embodiments, prostate cancer polypeptide levels are determined in vitro by measuring protein or mRNA levels. Protein levels are measured using immunoassay methods such as Western blotting, ELISA, etc. with antibodies that selectively bind to prostate cancer polypeptides or fragments thereof. For the measurement of mRNA, for example, PCR, amplification using LCR, or hybridization assay methods such as Northern hybridization, RNase protection and dot blotting are preferred. Protein or mRNA levels are detected using directly or indirectly labeled detection substances, such as fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies and the like herein. Described in.
[0265]
Alternatively, a reporter gene system can be devised using a prostate cancer protein promoter operably linked to a reporter gene such as luciferase, green fluorescent protein, CAT, or β-gal. This reporter construct is typically transfected into cells. After treatment with a potential modulator, the amount of transcription, translation or activity of the reporter gene is measured according to standard techniques known to those skilled in the art.
[0266]
In preferred embodiments, screening can be performed on individual genes and gene products (proteins) as outlined above. That is, a particular differentially expressed gene is identified as important in a particular state, and a modulator can be screened for the expression of the gene or gene product itself. The gene product of a differentially expressed gene is sometimes referred to herein as a “prostate cancer protein”. The prostate cancer protein can be a fragment or a full-length protein for the fragments shown herein.
[0267]
In certain embodiments, screening for modulators of specific gene expression is performed. Typically, the expression of only one or several genes is evaluated. In another embodiment, the screen is designed to find compounds that bind to the first differentially expressed protein. These compounds are then evaluated for their ability to modulate differentially expressed activity. Furthermore, once the first candidate compound has been identified, variants are further screened to better assess structure-activity relationships.
[0268]
In a preferred embodiment, a binding assay is performed. In general, purified or isolated gene products are used, ie, the gene product of one or more differentially expressed nucleic acids is made. For example, antibodies are made against the protein gene product and standard immunoassay methods are attempted to determine the amount of protein present. Alternatively, cells containing prostate cancer protein can be used in these assays.
[0269]
Thus, in a preferred embodiment, these methods comprise combining a prostate cancer protein with a candidate compound and determining the binding of this compound to the prostate cancer protein. Preferred embodiments utilize human prostate cancer proteins, although other mammalian proteins can be used, for example, in the development of animal models of human disease. In some embodiments, variant or derivative prostate cancer proteins can be used as outlined herein.
[0270]
In general, in a preferred embodiment of the methods herein, prostate cancer protein or candidate substance is non-diffusably transferred to an insoluble support having an independent sample receiving area (eg, microtiter plate, array, etc.). ) Combined. The insoluble support can be made from any composition to which the composition binds and is easily separated from soluble material, or otherwise compatible with the overall screening method. The surface of such a support can be solid or porous and can have any convenient shape. Examples of suitable insoluble supports are microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (eg, polystyrene), polysaccharides, nylon, or nitrocellulose, Teflon. Microtiter plates and arrays are particularly convenient because a very large number of assays can be performed simultaneously using small amounts of reagents and samples. The specific composition binding method is not critical as long as it is compatible with the reagents and methods of the invention in general, maintains the activity of the composition, and is non-diffusible. The preferred method of conjugation is the use of antibodies (when the protein is bound to the support, neither the ligand binding site nor the activation sequence sterically hinders), “sticky” or direct binding to the ionic support. , Chemical cross-linking, synthesis of proteins or substances on its surface, and the like. After protein or substance binding, excess unbound material is washed away. The sample receiving area can then be blocked by incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
[0271]
In a preferred embodiment, prostate cancer protein is bound to a support and a test compound is added to the assay. Alternatively, the candidate substance is bound to a support and prostate cancer protein is added. Novel binding agents include specific antibodies, non-natural binding agents identified in chemical library screening, peptide analogs, and the like. Of particular interest are screening assays for substances that have low toxicity for human cells. A wide variety of assays are available for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, protein binding immunoassays, functional assays (such as phosphorylation assays), etc. Can be used for
[0272]
Determining the binding of a test modulating compound to prostate cancer protein can be done in a number of ways. In preferred embodiments, the compound is labeled, for example, attaching all or part of a prostate cancer protein to a solid support, adding a labeled candidate substance (eg, a fluorescent label), washing away excess reagent, And the determination of whether the label is present on the solid support directly determines the binding. Various blocking and cleaning steps can be utilized as appropriate.
[0273]
In some embodiments, only one component can be labeled, for example, a protein (or proteinaceous candidate compound) can be labeled. Or more than one component, for example for proteins¹²⁵I and compounds can be labeled with different labels, such as fluorescent groups. Proximity reagents such as quenchers or energy transfer agents are also useful.
[0274]
In certain embodiments, test compound binding is determined by competitive binding assays. A competitor is a binding moiety that is known to bind to a target molecule (ie, prostate cancer protein), such as an antibody, peptide, binding partner, ligand, and the like. Under certain circumstances, the compound and the binding moiety bind competitively, and the binding moiety replaces the compound. In certain embodiments, the test compound is labeled. The compound, either the competitor, or both are first added to the protein for a time sufficient to allow binding if it is present. Incubations are carried out at temperatures that promote optimal activity, typically between 4 and 40 ° C. Incubation times are typically optimized to facilitate, for example, rapid high throughput screening. Typically 0.1 to 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, indicating binding according to the presence or absence of the labeled component.
[0275]
In a preferred embodiment, the competitor is added first, followed by the test compound. Competitive replacement is an indication that the test compound binds to prostate cancer protein, and thus is capable of binding to and possibly modulating prostate cancer protein activity. In this embodiment, any component can be labeled. Thus, for example, when the competing target is labeled, the presence of the label in the wash solution is an indication that this substance has been replaced. Alternatively, if the test compound is labeled, the presence of the label on the support is an indication of replacement.
[0276]
In an alternative embodiment, the test compound is added first, followed by competitors, incubated and washed. The absence of binding by the competitor may indicate that the test compound has bound to prostate cancer protein with high affinity. Thus, when a test compound is labeled, the presence of a label on the support that lacks competitor binding may indicate that the test compound is capable of binding to prostate cancer protein.
[0277]
In preferred embodiments, these methods include differential screening to identify agents that are capable of modulating the activity of prostate cancer proteins. In this embodiment, these methods comprise a combination of prostate cancer protein and a competitor in the first sample. The second sample includes a test compound, prostate cancer protein, and a competitor. Competitor binding is determined for both samples, and the change or difference in binding between the two samples is the presence of a substance capable of binding to prostate cancer protein and potentially modulating its activity. Is shown. That is, if the binding of the competitor is different in the second sample compared to the first sample, the substance can bind to the prostate cancer protein.
[0278]
Alternatively, differential screening is used to identify drug candidates that bind to native prostate cancer protein but not to modified prostate cancer protein. The structure of the prostate cancer protein has been modeled and used in theoretical drug design to synthesize substances that interact with that site. Drug candidates that affect the activity of prostate cancer protein are also identified by screening for drugs for either ability to enhance or reduce the activity of this protein.
[0279]
Positive and negative controls can be used in these assays. Preferably, the control sample and test sample are run in at least triplicate to obtain statistically significant results. All sample incubations are performed for a time sufficient for the substance to bind to the protein. Following incubation, the sample is washed free of non-specifically bound material, and the amount bound, generally the amount of labeled material, is determined. For example, if a radioactive label is used, the sample is counted in a scintillation counter to determine the amount of bound compound.
[0280]
A variety of other reagents can be included in the screening assay. These include reagents such as salts, neutral proteins such as albumin, surfactants, etc., to promote optimal protein-protein binding and / or reduce non-specific or background interactions. Used for. On the other hand, reagents that improve assay efficiency such as protease inhibitors, nuclease inhibitors, antimicrobial agents and the like can also be used. The mixture of components can be added in an order that provides the requisite binding.
[0281]
In a preferred embodiment, the present invention provides a method of screening for compounds that are capable of modulating the activity of prostate cancer proteins. These methods include adding a test compound, as described above, to cells containing prostate cancer protein. Preferred cell types include almost any cell. These cells contain a recombinant nucleic acid encoding a prostate cancer protein. In a preferred embodiment, the library of candidate substances is tested on a plurality of cells.
[0282]
In certain aspects, these assays are based on, for example, hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutic agents, radiation, carcinogens, or other cells (ie, cells Assess the presence or absence of physiological signals or previous or subsequent exposure, such as cell contact). In another example, these determinations are determined at different phases of cell cycle progression.
[0283]
In this method, compounds that modulate prostate cancer material are identified. Compounds with pharmacological activity can enhance or interfere with the activity of prostate cancer proteins. Once identified, similar structures are evaluated to determine important structural features of the compound.
[0284]
In certain embodiments, a method of inhibiting prostate cancer cell division is provided. This method involves administration of a prostate cancer inhibitor. In another aspect, a method for inhibiting prostate cancer is provided. This method involves administration of a prostate cancer inhibitor. In a further aspect, a method of treating a cell or individual with prostate cancer is provided. This method involves administration of a prostate cancer inhibitor.
[0285]
In certain embodiments, the prostate cancer inhibitor is an antibody as described above. In another embodiment, the prostate cancer inhibitor is an antisense molecule.
[0286]
As described below, various cell growth, proliferation and metastasis assays are known to those skilled in the art.
[0287]
Soft agar growth or colony formation in suspension
Normal cells require a solid substrate to adhere and grow. When cells are transformed, they lose this phenotype and grow away from the substrate. For example, transformed cells are grown in a stirring suspension culture or suspended in a semi-solid medium such as semi-solid or soft agar. When transformed cells are transfected with a tumor suppressor gene, this regenerates the normal phenotype and requires a solid substrate to adhere and grow. Colony formation in soft agar growth or suspension assays can be used to identify modulators of prostate cancer sequences that inhibit the growth and transformation of abnormal cells when expressed in host cells. The therapeutic compound will reduce or eliminate the ability of the host cells to grow in agitated suspension culture or suspended in a semi-solid medium such as semi-solid or soft.
[0288]
For the technique of colony formation in soft agar growth or suspension assays, see Freshney's “Culture of Animal Cells a Manual of Basic Technique” (3rd edition, 1994), This is incorporated herein by reference. See also the “Method” section of the article by Garkavtsev et al. (1996, supra), which is incorporated herein by reference.
[0289]
Contact inhibition of growth and density limit
Normal cells typically grow in a flat and directional pattern in a Petri dish until they come into contact with other cells. When cells come into contact with another cell, they are blocked from contact and stop growing. However, when cells are malignantly transformed, they are not contact blocked and continue to grow to high density in non-directional growth foci. Thus, malignant transformed cells proliferate to a higher saturation concentration than normal cells. This can be detected morphologically by forming a non-directional monolayer of cells or circular cells within the growth foci within a regular pattern of normal surrounding cells. Alternatively, at saturation density (³H) -Thymidine labeling index can be used to determine the growth density limit. See Freschney's paper (1994), supra. Malignantly transformed cells, when transfected with a tumor suppressor gene, will regenerate the normal phenotype, begin to be contact blocked, and grow to a lower density.
[0290]
In this assay, at saturation density (³The labeling index with H) -thymidine is a preferred method for measuring the growth density limit. Transformed host cells are transfected with sequences associated with prostate cancer and grown at saturating density for 24 hours in non-limiting medium conditions. (³The percentage of cell labeling with H) -thymidine is determined by autoradiography. See Freschney's paper (1994), supra.
[0291]
Growth factor or serum dependent
Malignantly transformed cells have lower serum dependence than their normal counterparts (eg, Temin, J. Natl. Cancer Insti, 37: 167-175 (1966); Eagle et al., J. Exp. Med. 131, 836-879 (1970)); Freshney, supra). This is due in part to the release of various growth factors by malignant transformed cells. The growth factor or serum dependence of the malignant transformed host cell can be compared to that of the control.
[0292]
Tumor-specific marker level
Tumor cells release an increased amount of certain factors (hereinafter “tumor specific markers”) over their normal counterparts. For example, plasminogen activator (PA) is released from human glioma at higher levels than normal brain cells (eg, Gullino, “Angiogenesis, Neoplastic Angiogenesis, and Potential Tumor Growth Inhibition). (Angiogenesis, tumor vascularization, and potential interference with tumor growth) ", Biological Responses in Cancer, pages 178-184, (Mich., 1985)). Similarly, neoplastic angiogenic factor (TAF) is released at higher levels in tumor cells than its normal counterpart. See, eg, Folkman, “Angiogenesis and Cancer”, Sem Cancer Biol. , (1992)).
[0293]
Various techniques for measuring the release of these factors are described in Freshney (1994), supra. Furthermore, Uncless et al. Biol. Chem. 249: 4295-4305 (1974); Strickland and Beers, J.A. Biol. Chem. 251: 5694-5702 (1976); Whur et al., Br. J. et al. Cancer, 42: 305-312 (1980); Gullino, "Angiogenesis, tumor angiogenesis, and potential interference with tumor growth, Biomass." in Cancer, pp. 178-184, (Mich, edited by 1985)); Freshney, Anticancer Res. 5: 111-130 (1985).
[0294]
Infiltration into Matrigel
The degree of invasion to Matrigel or some other extracellular matrix component can be used as an assay to identify compounds that modulate sequences associated with prostate cancer. Tumor cells show a good correlation between malignancy and cell invasion into Matrigel or some other extracellular matrix component. In this assay, tumorigenic cells are typically used as host cells. Expression of tumor suppressor genes in these host cells will reduce host cell infiltration.
[0295]
The techniques described in Freshney's paper (1994, supra) can be used. Briefly, the level of host cell invasion can be measured using a filter coated with Matrigel or several extracellular matrix components. Penetration into the gel or the distal side of the filter is graded as invasiveness, depending on the number of cells and distance traveled¹²⁵Pre-labeling of cells with I and histological grading by measuring radioactivity at the distal side of the filter or at the bottom of the dish. See, for example, Freschney's paper (1984, supra).
[0296]
Tumor growth in vivo
The effects of prostate cancer related sequences on cell growth can be tested in transgenic or immunosuppressed mice. Knockout transgenic mice can be created in which the prostate cancer gene has been disrupted or the prostate cancer gene has been inserted. Knockout transgenic mice can be created by insertion of a marker gene or other heterologous gene into the endogenous prostate cancer gene site of the mouse genome by homologous recombination. Such mice can also be generated by replacement of an endogenous prostate cancer gene with a mutated version of the prostate cancer gene or by mutation of an endogenous prostate cancer gene, for example by exposure to a carcinogen.
[0297]
The DNA construct is introduced into the nucleus of embryonic stem cells. Cells containing the newly engineered genetic lesion are injected into the host mouse embryo, which is re-implanted into the recipient female. Some of these embryos generate chimeric mice with germ cells derived in part from this mutant cell line. By breeding chimeric mice in this way, it is possible to obtain new strains of mice containing the introduced genetic lesion (see, eg, Capecchi et al., Science, 244: 1288 (1989)). Chimeric targeted mice are described in Hogan et al. ("Manipulating the Mouse Embryo: A Laboratory Manual", Cold Spring Harbor Laboratory (1988)) Embryonic Stem Cells: Practical Methods (Teratocarcinomas and Embryonic Stems Cells: A Practical Approach) ", edited by Robertson, IRL Press, Washington D.C. C. , (1987).
[0298]
Alternatively, a variety of immunosuppressed or immunodeficient host animals can be used. For example, genetic athymic “nude” mice (see, eg, Giovanella et al., J. Natl. Cancer Inst., 52: 921 (1974)), SCID mice, thymectomized mice, or irradiated mice (eg, Bradley et al., Br. J. Cancer, 38: 263 (1978); Selby et al., Br. J. Cancer, 41:52 (1980)) can be used as a host. Implantable tumor cells injected into isogenic hosts (typically about 10⁶Single cells) will form invasive tumors in a high proportion of cases, while normal cells of similar origin will not occur. In hosts that have developed invasive tumors, cells expressing sequences associated with prostate cancer are injected subcutaneously. After an appropriate period, preferably 4-8 weeks, tumor growth was measured (eg, by volume or bi-directional maximum dimension) and compared to controls. Tumors that show a statistically significant decrease (eg, using Student's T test) are referred to as growth inhibited.
[0299]
Methods for identifying sequences associated with variant prostate cancer
Although there is no theoretical support, the expression of various prostate cancer sequences is correlated with prostate cancer. Thus, disorders based on mutant or variant prostate cancer genes can be determined. In certain embodiments, the invention provides a method of identifying a cell containing a variant prostate cancer gene, eg, determining all or part of the sequence of at least one endogenous prostate cancer gene in the cell. This is accomplished using a number of sequencing techniques. In a preferred embodiment, the present invention provides a method for identifying an individual's prostate cancer genotype, eg, determining all or part of the sequence of at least one prostate cancer gene in the individual. This is generally performed in at least one tissue of the individual and can include evaluation of many tissues or different samples of the same tissue. The method includes comparing the sequence of the sequenced prostate cancer gene to a known prostate cancer gene, ie, a wild type gene.
[0300]
The sequence of all or part of the prostate cancer gene can then be compared with known prostate cancer gene sequences to determine if any differences exist. This can be done using any number of known homology programs, such as Bestfit. In preferred embodiments, the presence of a sequence difference between a patient's prostate cancer gene and a known prostate cancer gene correlates with a disease state or disease state trend, as outlined herein.
[0301]
In a preferred embodiment, the prostate cancer gene is used as a probe to determine the copy number of the prostate cancer gene in the genome.
[0302]
In another preferred embodiment, the prostate oncogene is used as a probe to determine chromosomal localization of the prostate oncogene. Information such as chromosomal localization finds use in providing diagnosis or prognosis, particularly when chromosomal abnormalities such as translocations are identified at the prostate cancer locus.
[0303]
Administration of pharmaceutical and vaccine compositions
In one embodiment, a therapeutically effective amount of prostate cancer protein or a modulator thereof is administered to the patient. As used herein, “therapeutically effective amount” means a dose that produces an effect on what is administered. The exact dose will depend on the therapeutic purpose and will be ascertainable by one skilled in the art using known techniques (eg, Ansel et al., “Pharmaceutical dosage forms and drug delivery”); Lieberman, “Pharmaceutical Dosage Forms (1-3, 1992), Dekker, ISBN0824747084, 0824769918X, 0824712692, 08247169981; Lloyd,“ Therapeutic Technology, Science and Technique Compounding "(1999); and Pickar," Dose Determination ( osage Calculations, (1999)) As known in the art, in addition to prostate cancer regression, systemic versus local delivery, and modulation of novel protease synthesis rates, age, weight, general condition, gender, The severity of the meal, time of administration, drug interaction and condition is necessary and can be ascertained by one of ordinary skill in the art by routine experimentation US Patent Application No. 09 / 687,576 further describes The use and methods of diagnostic and treatment compositions in prostate cancer are disclosed and are incorporated herein by reference.
[0304]
A “patient” for the purposes of the present invention includes both humans and other animals, particularly mammals. Therefore, these methods are applicable for both clinical treatment and veterinary applications. In preferred embodiments, the patient is a mammal, preferably a primate, and in the most preferred embodiment the patient is a human.
[0305]
As described above, administration of the prostate cancer proteins of the present invention and modulators thereof is oral, subcutaneous, intravenous, nasal, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, or intraocular. Administration can be accomplished in a variety of ways, including but not limited to administration. In some cases, such as treatment of wounds and inflammation, prostate cancer proteins and modulators can be applied directly as solutions or sprays.
[0306]
The pharmaceutical composition of the present invention contains prostate cancer protein in a form suitable for administration to a patient. In a preferred embodiment, the pharmaceutical composition is in a water-soluble form, eg, exists as a pharmaceutically acceptable salt, which is meant to include both acid and base addition salts. “Pharmaceutically acceptable acid addition salts” include salts that maintain the biological effectiveness of the free base, as well as inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and acetic acid, Propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene By non-biological or otherwise undesirable salts formed with organic acids such as sulfonic acid, salicylic acid and the like. “Pharmaceutically acceptable base addition salts” include salts derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary, secondary and tertiary amines, substituted amines including natural substituted amines, cyclic amines, and basic ion exchange resins such as isopropyl Included are salts of amines, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
[0307]
The pharmaceutical composition may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn starch and other starches; binding Agents; sweeteners and other flavoring agents; colorants; and polyethylene glycols.
[0308]
The pharmaceutical composition can be administered in various unit dosage forms depending on the method of administration. For example, unit dosage forms suitable for oral administration include, but are not limited to, powders, tablets, pills, capsules, and lozenges. It will be appreciated that prostate cancer protein modulators (eg, antibodies, antisense constructs, ribozymes, small organic molecules, etc.) must be protected from digestion when administered orally. This is typically by complexing with a composition to confer resistance to acid and enzymatic hydrolysis of these molecules, or in a suitable resistant carrier such as a liposome or protein barrier. This is achieved by either packaging of these molecules. Means of protecting substances from digestion are well known in the art.
[0309]
The administration composition will usually comprise a prostate cancer protein modulator dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline. These solutions are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional, well-known sterilization techniques. These compositions can contain pharmaceutically acceptable auxiliary substances necessary to approach physiological conditions, such as pH regulators and buffers, toxicity regulators, etc., for example sodium acetate. Sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The active ingredient concentration in these formulations can vary widely and is selected according to the specific mode of administration selected and the needs of the patient, mainly based on liquid volume, viscosity, weight, etc. (e.g., See “Remington's Pharmaceutical Science” (15th edition, 1980), and Goodman and Giliman, “The Pharmacological Basis of Therapeutics 96,” Ed. (Hard 19).
[0310]
Thus, a typical pharmaceutical composition for intravenous administration is about 0.1-10 mg per patient per day. Use a dose of 0.1 to a maximum of about 100 mg per patient per day, especially if the drug is administered to an isolated site and not into the bloodstream of a body cavity or organ lumen. Can do. Substantially higher doses are possible for topical administration. Actual methods for preparing parenterally administrable compositions include “Remington's Pharmaceutical Science” and Goodman and Gilman “The Pharmacological Basis of Therapeutics” described above, It will be known or apparent to those skilled in the art.
[0311]
These compositions comprising a prostate cancer protein modulator can be administered for therapeutic or prophylactic treatment. In therapeutic applications, the composition is administered to a patient suffering from a disease (eg, cancer) in an amount sufficient to cure or at least partially resist the disease and its complications. An amount adequate to accomplish this is defined as "therapeutically useful amount". The effective amount for this use will vary depending on the severity of the disease and the general health of the patient. Single or repeated administrations of the compositions are administered depending on the dosage and frequency required and patient tolerance. In any event, the composition must provide a sufficient amount of the substance of the invention to effectively treat the patient. An amount of a modulator that can prevent or delay the onset of cancer in a mammal is referred to as a “prophylactically effective amount”. The specific dosage required for prophylactic treatment will depend on the medical condition and history of the mammal, the specific cancer to be prevented, and other factors such as age, weight, sex, route of administration, efficacy, etc. . Such prophylactic treatment can be used, for example, in mammals that are already responsive to cancer to prevent cancer recurrence, or in mammals that are suspected of developing cancer with a significant likelihood.
[0312]
It will be appreciated that the prostate cancer protein modulating compounds of the invention can be administered alone or in combination with additional prostate cancer modulating compounds or other therapeutic agents such as other anti-cancer agents or treatments.
[0313]
In many embodiments, one or more nucleic acids, eg, a polynucleotide comprising a nucleic acid sequence shown in Tables 1-16, such as an antisense polynucleotide or ribozyme, will be introduced into a cell in vitro or in vivo. . The present invention provides methods, reagents, vectors, and useful for the expression of polypeptides and nucleic acids associated with prostate cancer using in vitro (cell-free), ex vivo or in vivo (cell or organism based) recombinant expression systems. Provide cells.
[0314]
The specific procedure used for introducing a nucleic acid into a host cell for expression of the protein or nucleic acid is specific to the application. A number of techniques are used for introducing foreign nucleotide sequences into host cells. These include calcium phosphate transfection, spheroplasts, electroporation, liposomes, microinjections, plasma vectors for the introduction of cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into host cells. ), Including the use of viral vectors and any other well-known methods (eg, Berger and Kimmel, “Guide to Molecular Cloning Techniques, Methods in Enzymology”, vol. 152) Berger), Ausubel et al., “Current Protocols” (1999 Addendum), and Sambrook et al., “Molecular Clonin. - A Laboratory Manual (Molecular Cloning - A Laboratory Manual) "(second edition, 1-3, pp. 1989)).
[0315]
In a preferred embodiment, prostate cancer proteins and modulators are administered as therapeutic agents and can be formulated as outlined above. Similarly, prostate cancer genes (including full length sequences, partial sequences, or regulatory sequences of prostate cancer coding regions) can be administered in gene therapy applications. These prostate cancer genes include either antisense use as gene therapy (ie, integration into the genome) or as an antisense composition, as will be appreciated by those skilled in the art.
[0316]
Prostate cancer polypeptides and polynucleotides can also be administered as vaccine compositions to stimulate HTL, CTL and antibody responses. Such vaccine compositions include, for example, lipidated peptides (see, eg, Vitiello, A. et al., J. Clin. Invest., 95: 341 (1995)), poly (DL-lactide-co- -Peptide compositions encapsulated in microlides ("PLG") microspheres (eg Eldridge et al., Molec. Immunol., 28: 287-294 (1991); Alonso et al., Vaccine, 12: 299-306 (1994)). Jones et al., Vaccine, 13: 675-681 (1995)), peptide compositions contained in immune stimulating complexes (ISCOMS) (eg, Takahashi et al., Nature, 344: 873-875 (1990); Hu et al., Clin Exp munol., 113: 235-243 (1998)), multi-antigen peptide system (MAP) (eg Tam, Proc. Natl. Acad Sci. USA, 85: 5409-5413 (1988); Tam, J. Immunol. Methods, 196: 17-32 (1996)), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus et al. "Concepts in vaccine development" (Kaufmann ed., Page 379, 1996); Chakrabarti et al., Nature, 320: 535 (1986); Hu et al., Nat. re, 320: 537 (1986); Kiney et al., AIDS Bio / Technology 4: 790 (1986); Top et al., J. Infect. Dis., 124: 148 (1971); Chanda et al., Virology, 175: 535 1990)), particles of viral or synthetic origin (eg, Kofler et al., J. Immunol. Methods, 192: 25 (1996); Eldridge et al., Sem. Hematol, 30:16 (1993); Falo et al., Nature Med., 7 : 649 (1995)), adjuvants (Warren et al., Annu. Rev. Immunol. 4: 369 (1986); Gupta et al., Vaccine, 11: 293 (1993)), liposomes (Reddy et al., J. Biol. Immunol. 148: 1585 (1992); Rock, Immunol. Today, 17: 131 (1996)), or naked or particle-absorbed cDNA (Ulmer et al., Science, 259: 1745 (1993); Robinson et al., Vaccine, 11: 957 (1993); Shiver et al., “Vaccine Development”. Concepts in vaccine development "(Kaufmann, 423, 1996); Cease and Berzofsky, Annu. Rev. Immunol., 12: 923 (1994), and Eldridge et al., 16: 93. )). Toxin targeted delivery techniques are also known as receptor-mediated targeting, and those from Avant Immunotherapeutics (Needham, Mass.) Can also be used.
[0317]
Vaccine compositions often include an adjuvant. Many adjuvants are used to protect antigens from rapid metabolism, such as aluminum hydroxide or mineral oil, and immune proteins such as lipid A, Bordetella pertussis or Mycobacterium tuberculosis-derived proteins. Contains substances designed as response stimulants. Certain adjuvants are commercially available, eg, Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck, Rahway, NJ); AS-2 (SmithKline Beecham) , Philadelphia, PA); aluminum hydroxide gel (alum) or aluminum salts such as aluminum phosphate; calcium, iron or zinc salts; insoluble suspensions of acylated tyrosine; acylated carbohydrates; There are cationically or anionically derived polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines such as GM-CSF, interleukin-2, interleukin-7, interleukin-12, and other growth factors can also be used as adjuvants.
[0318]
A vaccine can be administered as a nucleic acid composition such that DNA or RNA encoding one or more polypeptides, or fragments thereof, is administered to a patient. This method is described, for example, in US Pat. Nos. 5,580,859; 5,589,466; 5,804,566, in addition to Wolff et al., Science, 247: 1465 (1990). 739,118; 5,736,524; 5,679,647; WO 98/04720; and described in more detail below. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymer, peptide mediated) delivery, cationic lipid complexes, and particle mediated (“gene gun”) or pressure mediated There is mold delivery (see, eg, US Pat. No. 5,922,687).
[0319]
For the purpose of therapeutic or prophylactic immunization, the peptides of the invention can be expressed by viral vectors or bacterial vectors. Examples of expression vectors include attenuated viral hosts such as vaccinia or fowlpox. This method relates to the use of vaccinia virus as a vector for expressing a nucleotide sequence encoding, for example, a prostate cancer polypeptide or polypeptide fragment. Upon introduction into the host, this recombinant vaccinia virus expresses an immunogenic peptide, thereby eliciting an immune response. Vaccinia vectors and methods useful in immunization protocols are disclosed, for example, in US Pat. No. 4,722,848. Another vector is BCG (Bacil Calmette Guerin). BCG vectors are described in Stover et al., Nature, 351: 456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization, such as adeno-associated virus vectors and adeno-associated virus vectors, retrovirus vectors, Salmonella typhi vectors, detoxified Bacillus anthracis toxin vectors, etc. Will be apparent to those skilled in the art (eg, Shata et al., Mol Med Today, 6: 66-71 (2000); Shedlock et al., J Leukoc Biol, 68: 793-806 (2000); Hipp et al., In Vivo, 14: 571-85 (2000)).
[0320]
The use of genes as DNA vaccines is well known, which places a prostate cancer gene or a portion of a prostate cancer gene under the control of a regulatable or tissue-specific promoter for expression in prostate cancer patients Including that. The prostate cancer gene used in the DNA vaccine can encode the full length prostate cancer protein, but more preferably encodes a portion of the prostate cancer protein including a peptide derived from the prostate cancer protein. In certain embodiments, the patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a prostate cancer gene. For example, the ability of genes encoding prostate cancer-related genes or sequences encoding small fragments of prostate cancer proteins to be introduced into expression vectors to generate their immunogenic and cytotoxic T cell responses related to MHC class I To be tested. This approach provides for the generation of cytotoxic T cell responses against cells presenting antigens, including intracellular epitopes.
[0321]
In a preferred embodiment, the DNA vaccine comprises a gene encoding an adjuvant molecule with a DNA vaccine. Such adjuvant molecules include cytokines that enhance the immunogenic response to prostate cancer polypeptides encoded by the DNA vaccine. Additional or alternative adjuvants are available.
[0322]
In another preferred embodiment, the prostate cancer gene has found use in the creation of animal models of prostate cancer. If the identified prostate cancer gene is suppressed or attenuated in cancer tissue, gene therapy techniques such as antisense RNA against the prostate cancer gene will also reduce or suppress expression of this gene. Animal models of prostate cancer have found use in screening for modulators of sequences associated with prostate cancer or modulators of prostate cancer. Similarly, transgenic animal technology, including gene knockout technology, will result in the absence or increased expression of prostate cancer protein, for example as a result of homologous recombination with an appropriate gene targeting vector. If desired, tissue-specific expression or knockout of prostate cancer protein may be required.
[0323]
Prostate cancer protein may be overexpressed in prostate cancer. Thus, transgenic animals overexpressing prostate cancer protein can be generated. Depending on the desired level of expression, this transgene can be expressed using promoters of various strengths. In addition, the copy number of the incorporated transgene can be determined and compared to the determination of the expression level of the transgene. An animal produced by such a method has been found to be used as an animal model of prostate cancer and is useful in screening for a modulator for treating prostate cancer.
[0324]
Kit for use in diagnostic and / or prognostic applications
Kits are also provided by the present invention for use in the previously suggested diagnostic, research and therapeutic applications. For diagnostic and research applications, such kits may include any or all of the following: assay reagents, buffers, prostate cancer specific nucleic acids or antibodies, hybridization probes and / or primers, antisense polys. Nucleotides, ribozymes, dominant negative prostate cancer polypeptides or polynucleotides, small molecule inhibitors of sequences associated with prostate cancer, and the like. Therapeutic products may include sterile saline or other pharmaceutically acceptable emulsion and suspension bases.
[0325]
In addition, these kits are equipped with industrial materials including instructions (ie protocols) regarding the practice of the method of the invention. Instructional materials typically include written or printed materials, but are not limited to such. Any medium capable of storing such instructions and communicating them to the end user is encompassed by the present invention. Such media include, but are not limited to, electronic storage media (eg, magnetic disks, tapes, cartridges, chips), optical media (eg, CD ROM), and the like. Such media may include an address on the Internet that provides instructional material.
[0326]
The invention also provides kits for screening for modulators of sequences associated with prostate cancer. Such kits can be prepared from readily available materials and reagents. For example, such a kit may include one or more of the following materials: a polypeptide or polynucleotide associated with prostate cancer, a reaction tube, and instructions for testing for activity associated with prostate cancer. Optionally, the kit includes a biologically active prostate cancer protein. A wide variety of kits and components can be prepared according to the present invention, depending on the intended user of the kit and the specific needs of the user. Diagnosis is typically associated with the evaluation of multiple genes or products. These genes are likely to be selected based on correlation with important parameters of the disease that can be ascertained with historical or outcome data.
[0327]
Example
Example 1: Tissue preparation, labeling chip, and fingerprint
TRIzol Total from tissue samples using reagents RNA Purification
The sample weight was first measured. The tissue sample was homogenized using a homogenizer (eg, Polytron 3100) in 1 ml of TRIzol per 50 mg of tissue. The size of the generator / probe used was determined by the amount of sample. A generator that is too large for the amount of tissue to be homogenized results in sample loss and lower RNA yield. A relatively large generator (eg 20 mm) is suitable for tissue samples weighed above 0.6 g. The fill tube must not overflow. A 15 ml polypropylene tube (Falcon 2059) with a working volume of more than 2 ml and no more than 10 ml is suitable for homogenization.
[0328]
Tissues were stored frozen until homogenized. TRIzol was added directly to the frozen tissue prior to homogenization. After homogenization, the insoluble material was removed from the homogenate by centrifugation at 4500C for 15 minutes at 7500 xg in Sorvall Superspeed or 10 minutes at 12,000 xg in an Eppendorf centrifuge. The clear homogenate was then transferred to a new tube. Samples were frozen and stored at −60 to −70 ° C. for at least 1 month or continued to be purified.
[0329]
The next stage is phase separation. The homogenized sample was incubated for 5 minutes at room temperature. Thereafter, 0.2 ml of chloroform was added to 1 ml of the TRIzol reagent to the homogenization mixture. The tubes were sealed with caps and shaken vigorously by hand for 15 seconds (not vortexed). These samples were then incubated for 2-3 minutes at room temperature and then centrifuged at 6500 rpm for 30 minutes at 4 ° C. at Sorvall Superspeed.
[0330]
The next step is RNA precipitation. The aqueous phase was transferred to a new tube. If isolation of DNA or protein is desired, the organic phase can be secured. Thereafter, 0.5 ml of isopropyl alcohol was added to 1 ml of the TRIzol reagent used in the initial homogenization. These tubes were then sealed with caps and inverted and mixed. These samples were then incubated at room temperature for 10 minutes and centrifuged at 6500 rpm for 20 minutes at 4 ° C. in Sorvall.
[0331]
The RNA was then washed. The supernatant was poured off and the pellet was washed with cold 75% ethanol. 1 ml of 75% ethanol was used per ml of TRIzol reagent used in the initial homogenization. These tubes were sealed with caps and inverted several times to loosen the pellets without vortexing. They were then centrifuged at <8000 rpm (<7500 × g) for 5 minutes at 4 ° C.
[0332]
The RNA wash was decanted. The pellet was carefully transferred to an Eppendorf tube (the tube was slid down into a new tube using a pipette tip to help route it if necessary). The tube size for RNA precipitation was determined according to the working volume. Larger tubes take longer to dry. The pellet was dried. The RNA is then DEPC H₂Resuspended in an appropriate amount of O (eg 2-5 μg / μl). Thereafter, the absorbance was measured.
[0333]
Next, poly A + mRNA was purified from the total RNA by other methods such as Qiagen RNeasy kit. Poly A + mRNA was purified from total RNA by addition of an Oligotex suspension heated at 37 ° C. and mixing prior to addition to RNA. The elution buffer was incubated at 70 ° C. If there was a precipitate in the buffer, the 2x binding buffer was warmed to 65 ° C. Total RNA was mixed with DEPC-treated water, 2x binding buffer, and Oligotex as described in Table 2 on page 16 of the Oligotex Handbook, then incubated at 65 ° C for 3 minutes and at room temperature for 10 minutes.
[0334]
The preparation was centrifuged at 14,000-18,000 g for 2 minutes, preferably at the “soft setting”. The supernatant was removed without disturbing the Oligotex pellet. A small amount of solution was left to reduce the loss of oligotex. The supernatant was saved until satisfactory binding and elution of poly A + mRNA was observed.
[0335]
The preparation was then gently resuspended in wash buffer 0W2, pipetted onto a spin column and centrifuged for 1 minute at maximum speed (soft setting if possible).
[0336]
The spin column was then transferred to a new collection tube, gently resuspended in wash buffer 0W2, and centrifuged as described herein.
[0337]
The spin column was then transferred to a new tube and eluted with 20-100 μl of preheated (70 ° C.) elution buffer. Oligotex resin was gently resuspended by pipetting up and down. The above centrifugation was repeated and the elution was repeated with fresh elution buffer, or the first eluent was made to reduce the elution volume.
[0338]
The diluted elution buffer was then used as a blank and the absorbance was read to determine the yield.
[0339]
Before proceeding to cDNA synthesis, the remaining components or components in the elution buffer from the Oligotex purification procedure inhibited the enzymatic reaction downstream of the mRNA, so this mRNA was precipitated and then proceeded to cDNA synthesis. 0.4 volume of 7.5M NH₄OAc + 2.5 volumes of cold 100% ethanol is added and the preparation is allowed to precipitate at −20 ° C. for 1 hour to overnight (or at −70 ° C. for 20-30 minutes) and 30 at 14,000-16,000 × g. Centrifuged for 4 minutes at 4 ° C. The pellet was then washed with 0.5 ml of 80% ethanol (−20 ° C.) and then centrifuged at 14,000-16,000 × g for 5 minutes at room temperature. Next, washing with 80% ethanol was repeated. The last small amount of ethanol from the pellet is dried without using a speed vacuum and the pellet is then DEPC H₂Resuspended in O at a concentration of 1 μg / μl.
[0340]
Alternative alternative ( For example Qiagen Company RNeasy kit ) Use RNA Purification
It was added to the RNeasy column so that it did not exceed 100 μg. The sample volume was adjusted to 100 μl with RNase-free water. To the sample was added 350 μl buffer RLT followed by 250 μl ethanol (100%). The preparation was then mixed by pipetting and loaded onto an RNeasy mini spin column for centrifugation (> 10,000 rpm for 15 seconds). If the yield was low, the column was re-loaded with the flow-through and re-centrifuged.
[0341]
The column was then transferred to a new 2 ml collection tube, 500 μl buffer RPE was added and centrifuged at> 10,000 rpm for 15 seconds. The flow-through was discarded. 500 μl buffer RPE was then added and the preparation was centrifuged at> 10,000 rpm for 15 seconds. The flow-through was discarded and the column membrane was dried by centrifugation at maximum speed for 2 minutes. The column was transferred to a new 1.5 ml collection tube. 30-50 μl of RNase-free water was placed directly on the column membrane. The column was then centrifuged at> 10,000 rpm for 1 minute and the elution step was repeated.
[0342]
The absorbance was then read to determine the yield. If necessary, these materials can be ethanol precipitated with ammonium acetate and 2.5X volume of 100% ethanol.
[0343]
First strand cDNA Synthesis of
The first strand can be made using Gibco's “SuperScript Choice System for cDNA Synthesis” kit. Starting material is 5 μg of total RNA or 1 μg of poly A + mRNA. For total RNA, 2 μl of SuperScript RT was used; for poly A + mRNA, 1 μl of Superscript RT was used. The final volume of the first strand synthesis mixture was 20 μl. The RNA volume should not exceed 10 μl. RNA was incubated with 1 μl of 100 pmol T7-T24 oligo for 10 minutes at 70 ° C., then 7 μl was added on ice: 4 μl 5 × first strand buffer, 2 μl 0.1 M DTT, and 1 μl 10 mM dNTP mixture. This preparation was then incubated at 37 ° C. for 2 minutes, then added to Superscript RT and incubated at 37 ° C. for 1 hour.
[0344]
Second strand synthesis
For second strand synthesis, the first strand reaction was placed on ice and the following was added: 91 μl DEPC H₂O; 30 μl 5X second strand buffer; 3 μl 10 mM dNTP mix; 1 μl 10 U / μl E. coli DNA ligase; 4 μl 10 U / μl E. coli DNA polymerase; and 1 μl 2 U / μl RNase H. Mixing and incubation were performed at 16 ° C. for 2 hours. 2 μl of T4 DNA polymerase was added. Incubated at 16 ° C. for 5 minutes. 10 μl of 0.5M EDTA was added.
[0345]
cDNA Purification
The cDNA was purified using phenol: chloroform: isoamyl alcohol (25: 24: 1) and Phase-Lock gel tubes. These PLG tubes were centrifuged at maximum speed for 30 seconds. The cDNA mixture was then transferred to a PLG tube. An equal volume of phenol: chloroform: isoamyl alcohol was added and the preparation was shaken vigorously (not vortexed) and centrifuged at maximum speed for 5 minutes. Transfer the top aqueous solution to a new tube and add 7.5x 5M NH₄Ethanol was precipitated by addition of OAc and 2.5x volume of 100% ethanol. Immediately thereafter, it was centrifuged at the highest speed for 20 minutes at room temperature. The supernatant was removed and the pellet was washed with 2x cold 80% ethanol. As much ethanol wash as possible was removed before air drying of the pellet; resuspended in 3 μl RNase-free water.
[0346]
In vitro transcription (IVT) And biotin labeling
In vitro transcription (IVT) and biotin labeling were performed as follows: pipetting 1.5 μl of cDNA into thin wall PCR tubes. The NTP labeling mixture was made up of 2 μl T7 10 × ATP (75 mM) (Ambion); 2 μl T7 10 × GTP (75 mM) (Ambion); 1.5 μl T7 10 × CTP (75 mM) (Ambion); 1.5 μl T7 10 × UTP (75 mM) (Ambion) 3.75 μl 10 mM Bio-11-UTP (Boehringer-Mannheim / Roche or Enzo); 3.75 μl 10 mM Bio-16-CTP (Enzo); 2 μl 10 × T7 transcription buffer (Ambion); and 2 μl 10 × T7 enzyme mixture It depends on the combination of (Ambion). The final volume was 20 μl. Incubated for 6 hours at 37 ° C. in a PCR machine. The RNA was further purified. Purification was according to previous instructions for RNeas column or Qiagen RNeasy protocol handbook. cRNA often requires ethanol precipitation by resuspension in a volume compatible with the fragmentation step.
[0347]
Fragmentation was performed as follows. 15 μg of labeled RNA was fragmented normally. To attempt to minimize the fragmentation reaction volume, a 10 μl volume is recommended, but 20 μl may be used. Magnesium present in the fragmentation buffer contributes to precipitation of the hybridization buffer and should not exceed 20 μl. RNA fragmentation is by incubation at 94 ° C. for 35 minutes in 1 × fragmentation buffer (5 × fragmentation buffer is 200 mM Tris acetic acid, pH 8.1; 500 mM KOAc; 150 mM MgOAc). Labeled RNA transcripts can be analyzed before and after fragmentation. Samples were heated at 65 ° C. for 15 minutes and electrophoresed on a 1% agarose / TBE gel to give an approximate estimate of the transcript size range.
[0348]
For hybridization, 200 μl of hybridization mixture (10 μg cRNA) was placed on the chip. If multiple rounds of hybridization are to be performed (eg cycling with 5 chipsets), it is recommended that the initial hybridization mix be made up to 300 μl or more. The hybridization mix is shown below: labeled RNA fragment (50 ng / μl final concentrate); 50 pM 948-b control oligo; 1.5 pM BioB; 5 pM BioC; 25 pM BioD; 100 pM CRE; 0.1 mg / ml herring Sperm DNA; 0.5 mg / ml acetylated BSA; and 300 μl of 1 × MES hyb buffer.
[0349]
The hybridization reaction was performed with non-biotinylated IVT (purified by RNeasy column) (see Example 1 for IVT step from tissue): The following mixture was prepared:

The 14 μl mixture was incubated at 70 ° C. for 10 minutes and then placed on ice.
[0350]
The following mixture was used for the reverse transcription method:

The solution was added to the hybridization reaction solution and incubated at 42 ° C. for 30 minutes. Thereafter, 1 μl of SSII was added, further incubated for 1 hour, and then placed on ice.
[0351]
The 50 × dNTP mixture contains 25 mM cold dATP, dCTP, and dGTP, 10 mM dTTP, and 25 μl each of 100 mM dATP, dCTP, and dGTP; 10 μl of 100 mM dTTP,₂Prepared by adding to 15 μl of O.
[0352]
RNA degradation was performed as follows. 86 μl of H₂To O, 1.5 μl 1M NaOH / 2 mM EDTA was added and incubated at 65 ° C. for 10 minutes. For U-Con 30, 500 μl TE / sample was spun at 7000 g for 10 minutes and a flow-through was used for purification. For Qiagen purification, u-con recovered material was suspended in 500 μl buffer PB and proceeded using the Qiagen protocol. For DNAse digestion, 1 μl of 1/100 dilution of DNAse / 30 μl Rx was added and incubated at 37 ° C. for 15 minutes. DNAse was denatured by incubation at 95 ° C. for 5 minutes.
[0353]
Sample preparation
For sample preparation, add Cot-1 DNA, 10 μl; 50 × dNTP, 1 μl; 20 × SSC, 2.3 μl; sodium pyrophosphate, 7.5 μl; 10 mg / ml herring sperm DNA; 1 μl of 1/10 dilution, The final volume was 21.8. Dry under high speed vacuum. H₂Resuspended in 15 μl of O. 0.38 μl of 10% SDS was added. The mixture was heated at 95 ° C. for 2 minutes and slowly cooled to room temperature over 20 minutes. Placed on slide and hybridized overnight at 64 ° C. Washed after hybridization: 3 × SSC / 0.03% SDS: 2 minutes, H₂O 2x SSC 37.5 ml in 250 ml + 0.75 ml of 10% SDS; 1x SSC: 5 minutes, H₂O 20xSSC 12.5ml in 250ml; 0.2xSSC: 5 minutes, H₂O 250 ml 20x SSC in 250 ml. Slides were dried and scanned with appropriate PMTs and channels.
[0354]
Example 2: Taxol-resistant xenograft model of human prostate cancer
Therapeutic medication management including paclitaxel (Taxol; Bristol-Myers Squibb, Princeton, NJ) has been particularly successful in the treatment of phase II clinical trials of hormone refractory prostate cancer (Smith et al., Semin. Oncol., 26 (1 Suppl 2): 109-11 (1999)). However, many patients develop tumors that become taxol resistant first or later. The following experiments were conducted to identify genes associated with resistance to taxol, or genes that can modulate the response to taxol resistance and thus can be used for therapy, or taxol resistance in patients.
[0355]
The androgen-independent human cell line CWR22R was grown as a xenograft in nude mice (Nagabhushan et al., Cancer Res., 56 (13): 3042-4046 (1996); Agus et al., J. Natl. Cancer Inst., 91 (21): 1869-1876 (1999); Bubendorf et al., J. Natl. Cancer Inst., 91 (20): 1758-1764 (1999)). Initially, these xenograft tumors were sensitive to therapeutic doses of taxol. The mice were treated continuously at sub-therapeutic doses and the tumors were allowed to grow for 3-4 weeks before the tumors were surgically removed. Tumors from individual mice were then chopped and small portions were injected into healthy nude mice to establish the second passage of tumors. The mice were then continuously treated with an amount of taxol below the same therapeutic dose. This process was repeated 14 times to collect a portion of each generation of xenograft tumors. With each generation, resistance to therapeutic doses of taxol increased. By the end of this process, the tumor was completely resistant to therapeutic doses of taxol. RNA from each generation of tumors is then isolated, and individual mRNA species are probed to interrogate approximately 35,000 unique mRNA transcripts, with custom Affymetrix GeneChip® oligonucleotide microarrays. And quantified. Genes that showed statistically significant up-regulation or down-regulation in subsequent passages of increasing taxol-resistant tumors were selected. Only one gene was significantly upregulated, whereas 24 genes were downregulated. These are shown in Table 10.
[0356]
Gene sequences identified as being overexpressed in prostate cancer were used to identify coding regions from public DNA databases. These sequences are used to identify genes encoding known proteins, or they are exon estimation algorithms such as FGENESH (Salamov and Solovyev, Genome Res., 10: 516-522 (2000)). And used to estimate the coding region from genomic DNA. In addition, one of ordinary skill in the art would understand how to obtain unigene cluster identification and sequence information according to the typical accession numbers provided in Tables 1-16 (http: // www. ncbi.nlm.nih.gov/UniGene/).
[0357]
Table 1 shows genes that contain an expressed sequence tag and that are differentially expressed in prostate tumor tissue compared to normal tissue when analyzed using the Affymetrix / Eos Hu01 GeneChip array. The relative amount of each gene expressed in prostate tumor samples showing the highest gene expression and in various normal tissue samples is shown.

[0358]
Table 1A shows the deposit numbers of these prime keys that lack the unigeneID of Table 1. For each probe set, the inventors listed gene cluster numbers from which oligonucleotides were designed. Gene clusters were tabulated using Genbank EST and mRNA derived sequences. These sequences were clustered based on sequence similarity using a clustering and alignment tool (Clustering and Alignment Tools, Double Twist, Oakland, Calif.). The Genbank deposit number of the sequence containing each cluster is listed in the “Accession” column.

[0359]
Table 2 shows a preferred subset of the deposit numbers of the genes shown in Table 1 that were differentially expressed in prostate tumor tissue compared to normal prostate tissue.

[0360]
Table 2A shows the deposit numbers of these prime keys lacking the unigeneID in Table 2. For each probe set, the inventors listed gene cluster numbers from which oligonucleotides were designed. Gene clusters were tabulated using Genbank EST and mRNA derived sequences. These sequences were clustered based on sequence similarity using a clustering and alignment tool (Clustering and Alignment Tools, Double Twist, Oakland, Calif.). The Genbank deposit number of the sequence containing each cluster is listed in the “Accession” column.

[0361]
Table 3 shows genes that contain an expressed sequence tag and that are differentially expressed in prostate tumor tissue compared to normal tissue when analyzed using the Affymetrix / Eos Hu02 GeneChip array. The relative amount of each gene expressed in prostate tumor samples showing the highest gene expression and in various normal tissue samples is shown.

[0362]
Table 3A shows the deposit numbers of these prime keys lacking the unigeneID in Table 3. For each probe set, the inventors listed gene cluster numbers from which oligonucleotides were designed. Gene clusters were tabulated using Genbank EST and mRNA derived sequences. These sequences were clustered based on sequence similarity using a clustering and alignment tool (Clustering and Alignment Tools, Double Twist, Oakland, CA). The Genbank deposit number of the sequence containing each cluster is listed in the “Accession” column.

[0363]
Table 3B shows the genomic location of these prime keys lacking unigeneID and the deposit number in Table 3. For each estimated exon, we listed the source of the genomic sequence used for the estimation. The nucleotide position of each estimated exon is also listed.

[0364]
Table 4 shows a preferred subset of the deposit numbers of the genes found in Table 3 that are differentially expressed in prostate tumor tissue compared to normal prostate tissue.

[0365]
Table 4A shows the deposit numbers of these prime keys lacking the unigeneID in Table 4. For each probe set, the inventors listed gene cluster numbers from which oligonucleotides were designed. Gene clusters were tabulated using Genbank EST and mRNA derived sequences. These sequences were clustered based on sequence similarity using a clustering and alignment tool (Clustering and Alignment Tools, Double Twist, Oakland, Calif.). The Genbank deposit number of the sequence containing each cluster is listed in the “Accession” column.

[0366]
Table 4B shows the genomic location and deposit number of these prime keys lacking the unigeneID of Table 4. For each estimated exon, we listed the source of the genomic sequence used for the estimation. The nucleotide position of each estimated exon is also listed.

[0367]
Table 5 1170 genes up-regulated in prostate cancer compared to normal adult tissues
Table 5 shows 1170 genes that are up-regulated in prostate cancer compared to normal adult tissues. These were selected from 59680 probe sets on the Affymetrix / Eos Hu03 GeneChip array and the ratio of “average” prostate cancer to “average” normal adult tissue was greater than or equal to 3.44. The “average” prostate cancer level was set at the 85th percentile value in 73 prostate cancers. The “average” normal adult tissue level was set at the 85th percentile value among 162 non-malignant tissues. To remove gene-specific background levels of non-specific hybridization, the 7.5th percentile value in 162 non-malignant tissues was subtracted from both the numerator and denominator and then evaluated for this ratio.

[0368]
Table 5A shows the deposit numbers of these prime keys lacking the unigeneID of Tables 5, 6 and 7. For each probe set, the inventors listed the gene cluster number from which the oligonucleotide was designed. Gene clusters were tabulated using Genbank EST and mRNA derived sequences. These sequences were clustered based on sequence similarity using a clustering and alignment tool (Clustering and Alignment Tools, Double Twist, Oakland, Calif.). The Genbank deposit number of the sequence containing each cluster is listed in the “Accession” column.

[0369]
Table 5B shows the genomic location and deposit number of these prime keys lacking the unigeneID of Tables 5, 6 and 7. For each estimated exon, we listed the source of the genomic sequence used for the estimation. The nucleotide position of each estimated exon is also listed.

[0370]
Table 6 286 genes encoding extracellular or cell surface proteins that are up-regulated in prostate cancer compared to normal adult tissues
Table 6 shows 286 genes that are up-regulated in prostate cancer compared to normal adult tissue, possibly an extracellular or cell surface protein. These were selected as for Table 5 and the deduced protein contained a structural domain (eg, egf, 7tm domain) that is indicative of extracellular localization.

[0371]
Table 7. 42 genes encoding small molecule targets that are up-regulated in prostate cancer compared to normal adult tissues
Table 7 shows 42 genes that were up-regulated in prostate cancer compared to normal adult tissue, probably a small molecule target. These were selected as for Table 5 and the deduced proteins included structural domains (eg, proteases, kinases, phosphatase receptors) that are indicative of druggable structures. Functional domains are shown for each gene.

[0372]
Table 8 136 genes significantly down-regulated in prostate cancer compared to normal adult tissues
Table 8 shows 136 genes that were significantly down-regulated in prostate cancer compared to normal adult tissues. These were selected from 59680 probe sets on the Affymetrix / Eos Hu03 GeneChip array and the ratio of “mean” normal prostate to “mean” prostate cancer tissue was greater than or equal to 2. The “average” normal prostate level was set to the average of 4 normal prostate tissues. The “average” prostate cancer level was set at the 85th percentile value in 73 prostate cancers. To remove gene-specific background levels of nonspecific hybridization, the 10th percentile value in all tissues was subtracted from both the numerator and denominator and then evaluated for this ratio.

[0373]
Table 8A shows the deposit numbers of these prime keys lacking the unigeneID in Table 8. For each probe set, the inventors listed gene cluster numbers from which oligonucleotides were designed. Gene clusters were tabulated using Genbank EST and mRNA derived sequences. These sequences were clustered based on sequence similarity using a clustering and alignment tool (Clustering and Alignment Tools, Double Twist, Oakland, Calif.). The Genbank deposit number of the sequence containing each cluster is listed in the “Accession” column.

[0374]
Table 8B shows the genomic location and deposit number of these prime keys lacking the unigeneID in Table 8. For each estimated exon, we listed the source of the genomic sequence used for the estimation. The nucleotide position of each estimated exon is also listed.

[0375]
Table 9: 1001 genes significantly up-regulated in prostate gland compared to prostate cancer
Table 9 shows 1001 genes that were significantly upregulated in prostate cancer compared to normal prostate. These were selected from 59680 probe sets on the Affymetrix / Eos Hu03 GeneChip array and the ratio of “mean” normal prostate to “mean” prostate cancer tissue was greater than or equal to 8.14. The “average” normal prostate level was set to the average of 4 normal prostate tissues. The “average” prostate cancer level was set at the 85th percentile value in 73 tumor samples. To remove gene-specific background levels of nonspecific hybridization, the 10th percentile value in all tissues was subtracted from both the numerator and denominator and then evaluated for this ratio.

[0376]
Table 9A shows the deposit numbers of these prime keys lacking the unigeneID in Table 9. For each probe set, the inventors listed gene cluster numbers from which oligonucleotides were designed. Gene clusters were tabulated using Genbank EST and mRNA derived sequences. These sequences were clustered based on sequence similarity using a clustering and alignment tool (Clustering and Alignment Tools, Double Twist, Oakland, Calif.). The Genbank deposit number of the sequence containing each cluster is listed in the “Accession” column.

[0377]
Table 9B shows the genomic location and deposit number of these prime keys lacking the unigeneID in Table 9. For each estimated exon, we listed the source of the genomic sequence used for the estimation. The nucleotide position of each estimated exon is also listed.

[0378]
FIG. 10 shows genes comprising expressed sequence tags that are differentially expressed in taxol-resistant prostate tumor xenografts compared to taxol-sensitive prostate tumor xenografts. These genes have been shown to be either up-regulated or down-regulated while taxol resistance is induced during serial passage of the graft.

[0379]
Table 11 shows genes with expressed sequence tags that were up-regulated in prostate tumor tissue compared to normal prostate tissue when analyzed using the Affymetrix / Eos Hu01 GeneChip array. The ratio of “average” normal prostate to “average” prostate cancer tissue is shown.

[0380]
Table 11A shows the deposit numbers of these prime keys lacking the unigeneID in Table 11. For each probe set, the inventors listed gene cluster numbers from which oligonucleotides were designed. Gene clusters were tabulated using Genbank EST and mRNA derived sequences. These sequences were clustered based on sequence similarity using a clustering and alignment tool (Clustering and Alignment Tools, Double Twist, Oakland, CA). The Genbank deposit number of the sequence containing each cluster is listed in the “Accession” column.

[0381]
Table 12 shows genes with expressed sequence tags that were down-regulated in prostate tumor tissue compared to normal prostate tissue when analyzed using the Affymetrix / Eos Hu01 GeneChip array. The ratio of “average” normal prostate to “average” prostate cancer tissue is shown.

[0382]
Table 12A shows the deposit numbers of these prime keys lacking the unigeneID in Table 12. For each probe set, the inventors listed gene cluster numbers from which oligonucleotides were designed. Gene clusters were tabulated using Genbank EST and mRNA derived sequences. These sequences were clustered based on sequence similarity using a clustering and alignment tool (Clustering and Alignment Tools, Double Twist, Oakland, CA). The Genbank deposit number of the sequence containing each cluster is listed in the “Accession” column.

[0383]
Table 13 shows genes with expressed sequence tags that are up-regulated in prostate tumor tissue compared to normal prostate tissue when analyzed using the Affymetrix / Eos Hu02 GeneChip array. The ratio of “average” normal prostate to “average” prostate cancer tissue is shown.

[0384]
Table 13A shows the deposit numbers of these prime keys lacking the unigeneID in Table 13. For each probe set, the inventors listed gene cluster numbers from which oligonucleotides were designed. Gene clusters were tabulated using Genbank EST and mRNA derived sequences. These sequences were clustered based on sequence similarity using a clustering and alignment tool (Clustering and Alignment Tools, Double Twist, Oakland, Calif.). The Genbank deposit number of the sequence containing each cluster is listed in the “Accession” column.

[0385]
Table 13B shows the genomic location and deposit number of these prime keys lacking the unigeneID of Table 13. For each estimated exon, we listed the source of the genomic sequence used for the estimation. The nucleotide position of each estimated exon is also listed.

[0386]
Table 14 shows genes with expressed sequence tags that are down-regulated in prostate tumor tissue compared to normal prostate tissue when analyzed using the Affymetrix / Eos Hu02 GeneChip array. The ratio of “average” normal prostate to “average” prostate cancer tissue is shown.

[0387]
Table 14A shows the deposit numbers of these prime keys lacking the unigeneID in Table 14. For each probe set, the inventors listed gene cluster numbers from which oligonucleotides were designed. Gene clusters were tabulated using Genbank EST and mRNA derived sequences. These sequences were clustered based on sequence similarity using a clustering and alignment tool (Clustering and Alignment Tools, Double Twist, Oakland, Calif.). The Genbank deposit number of the sequence containing each cluster is listed in the “Accession” column.

[0388]
Table 14B shows the genomic location and deposit number of these prime keys lacking the unigeneID of Table 14. For each estimated exon, we listed the genomic sequence source used for the estimation. The nucleotide position of each estimated exon is also listed.

[0389]
Table 15 169 genes with sequence information shown in Table 16
Table 15 shows the unigene ID, unigene title, prime key, estimated cell localization, and typical deposit number for all the sequences in Table 16. The information in Table 15 is linked to EosCode in Table 16.

[0390]
Table 15A shows the deposit numbers of these prime keys lacking the unigeneID in Table 15. For each probe set, the inventors listed gene cluster numbers from which oligonucleotides were designed. Gene clusters were tabulated using Genbank EST and mRNA derived sequences. These sequences were clustered based on sequence similarity using a clustering and alignment tool (Clustering and Alignment Tools, Double Twist, Oakland, CA). The Genbank deposit number of the sequence containing each cluster is listed in the “Accession” column.

[0390]
Table 15B shows the genomic location and deposit number of these prime keys lacking the unigeneID of Table 15. For each estimated exon, we listed the genomic sequence source used for the estimation. The nucleotide position of each estimated exon is also listed.

[0392]
Table 11 and Sequence Listing

[0393]
It will be understood that the embodiments described above are not utilized in any manner to limit the true scope of the invention, but rather are presented for purposes of illustration. All publications, sequence accession numbers, and patent specifications cited herein are hereby incorporated by reference, as specifically and individually indicated that each publication or patent application is incorporated by reference. Incorporated into the book as a reference.

Claims (70)

A polynucleotide for the detection of transcripts associated with prostate cancer in a patient's cells, wherein the biological sample from the patient selectively hybridizes to a sequence at least 80% identical to the sequence shown in Tables 1-16 A method comprising the step of contacting with. The method of claim 1, wherein the polynucleotide selectively hybridizes to a sequence that is at least 95% identical to a sequence shown in Tables 1-16. The method of claim 1, wherein the biological sample is a tissue sample. The method of claim 1, wherein the biological sample comprises isolated nucleic acid. The method according to claim 4, wherein the nucleic acid is mRNA. 5. The method of claim 4, further comprising amplifying the nucleic acid prior to contacting the biological sample with the polynucleotide. The method of claim 1, wherein the polynucleotide comprises a sequence shown in Tables 1-16. The method of claim 1, wherein the polynucleotide is labeled. The method of claim 8, wherein the label is a fluorescent label. The method of claim 1, wherein the polynucleotide is immobilized on a solid surface. The method of claim 1, wherein the patient is undergoing therapeutic medication management to treat prostate cancer. The method of claim 1, wherein the patient is suspected of having prostate cancer. A method of monitoring the efficacy of therapeutic treatment of prostate cancer,
(I) procuring a biological sample from a patient undergoing therapeutic treatment; and (ii) selectively hybridizing the biological sample to a sequence that is at least 80% identical to the sequences shown in Tables 1-16. Contacting the soy polynucleotide, determining the level of transcript associated with prostate cancer in the biological sample, and thereby monitoring the efficacy of the therapy. (Iii) further comprising comparing the level of transcript associated with prostate cancer to the level of transcript associated with prostate cancer in a biological sample from a patient prior to or prior to therapeutic treatment. Item 14. The method according to Item 13. 14. The method of claim 13, wherein the patient is a human. A method of monitoring the efficacy of therapeutic treatment of prostate cancer,
(I) procuring a biological sample from a patient undergoing therapeutic treatment; and (ii) selectively hybridizing the biological sample to a sequence that is at least 80% identical to the sequences shown in Tables 1-16. Contacting a polypeptide encoded by a soy polynucleotide, wherein the polypeptide specifically determines a level of prostate cancer-related antibody in a biological sample that specifically binds to an antibody associated with prostate cancer, thereby Monitoring the efficacy of this therapy. And (iii) comparing the level of antibody associated with prostate cancer with the level of antibody associated with prostate cancer in a biological sample from a patient prior to or at an early stage of therapeutic treatment. The method described. The method of claim 16, wherein the patient is a human. A method of monitoring the efficacy of therapeutic treatment of prostate cancer,
(I) procuring a biological sample from a patient undergoing therapeutic treatment; and (ii) contacting the biological sample with an antibody, wherein the antibody is at least one of the sequences shown in Tables 1-16 and Determine the level of polypeptide associated with prostate cancer in a biological sample that specifically binds to a polypeptide encoded by a polynucleotide that selectively hybridizes to a sequence that is 80% identical. A method comprising the step of monitoring efficacy. (Iii) further comprising comparing the level of polypeptide associated with prostate cancer with the level of polypeptide associated with prostate cancer in a biological sample of a patient prior to or at an early stage of therapeutic treatment. 19. The method according to 19. 21. The method of claim 19, wherein the patient is a human. An isolated nucleic acid molecule consisting of the polynucleotide sequences shown in Tables 1-16. 23. The nucleic acid molecule of claim 22, wherein the nucleic acid molecule is labeled. 24. The nucleic acid of claim 23, wherein the label is a fluorescent label. An expression vector comprising the nucleic acid according to claim 22. A host cell comprising the expression vector of claim 25. An isolated polypeptide encoded by a nucleic acid molecule having the polynucleotide sequence shown in Tables 1-16. An antibody that specifically binds to the polypeptide of claim 27. 30. The antibody of claim 28, further conjugated to an effector component. 30. The antibody of claim 29, wherein the effector component is a fluorescent label. 30. The antibody of claim 29, wherein the effector component is a radioisotope or a cytotoxic chemical. 30. The antibody of claim 29, which is an antibody fragment. 30. The antibody of claim 29, wherein the antibody is a humanized antibody. 30. A method of detecting prostate cancer cells in a biological sample from a patient, comprising contacting the biological sample with an antibody of claim 28. 35. The method of claim 34, wherein the antibody is further conjugated to an effector component. 36. The method of claim 35, wherein the effector component is a fluorescent label. A method of detecting an antibody specific for prostate cancer in a patient, comprising contacting a biological sample from the patient with a polypeptide encoded by a nucleic acid comprising a sequence from Tables 1-16. A method of identifying a compound that modulates a polypeptide associated with prostate cancer,
(I) contacting the compound with a polypeptide associated with prostate cancer, wherein the polypeptide is encoded by a polynucleotide that selectively hybridizes to a sequence that is at least 80% identical to a sequence shown in Tables 1-16. And (ii) determining the functional effect of the compound on the polypeptide. 40. The method of claim 38, wherein the functional action is a physical action. 40. The method of claim 38, wherein the functional action is a chemical action. 40. The method of claim 38, wherein the polypeptide is expressed in a eukaryotic host cell or cell membrane. 40. The method of claim 38, wherein the functional effect is determined by measuring a ligand that binds to the polypeptide. 40. The method of claim 38, wherein the polypeptide is recombinant. 39. A method for inhibiting proliferation of cells associated with prostate cancer to treat prostate cancer in a patient, comprising administering to the subject a therapeutically effective amount of a compound identified using the method of claim 38. Including methods. 45. The method of claim 44, wherein the compound is an antibody. 46. The method of claim 45, wherein the patient is a human. (I) administering a test compound to a mammal having prostate cancer or cells isolated therefrom;
(Ii) the level of gene expression of a polynucleotide that selectively hybridizes to a sequence that is at least 80% identical to the sequence shown in Tables 1-16 in the treated cell or mammal; A drug screening assay, wherein the test compound that modulates the level of expression of the polynucleotide is a candidate for prostate cancer treatment. 48. The assay of claim 47, wherein the control is a mammal with prostate cancer or cells therefrom that have not been treated with a test compound. 48. The assay of claim 47, wherein the control is a normal cell or a mammal. 48. A method of treating a mammal having prostate cancer comprising administering a compound identified by the assay of claim 47. 48. A pharmaceutical composition for treating a mammal having prostate cancer, comprising a compound identified by the assay method of claim 47 and a physiologically acceptable excipient. A biological polynucleotide wherein the first polynucleotide selectively hybridizes to a sequence at least 80% identical to the first sequence shown in Tables 1-16; and at least a second sequence shown in Tables 1-16 The method of claim 1, wherein the method comprises contacting a plurality of polynucleotides comprising a second polynucleotide that selectively hybridizes to a sequence that is 80% identical. 53. The method of claim 52, wherein the plurality of polynucleotides comprises a third polynucleotide that selectively hybridizes to a sequence that is at least 80% identical to the third sequence shown in Tables 1-16. A method of detecting a transcript associated with prostate cancer, wherein a biological sample from a patient is contacted with a plurality of polynucleotides, wherein at least two of the polynucleotides have at least the sequences shown in Tables 1-16 and Selectively hybridizing to different sequences that are 80% identical. A method of detecting prostate cancer,
(I) procuring a biological sample from a patient;
(Ii) To determine the level of transcript associated with prostate cancer in a biological sample, the biological sample is selectively selected to a sequence that is at least 80% identical to the first sequence shown in Tables 1-16. A second sequence that is contacted with the first hybridizing polynucleotide; and at least 80% identical to a sequence not shown in Tables 1-16 to determine the expression level of the control transcript in the biological sample Contacting a second polynucleotide that selectively hybridizes to; wherein expression of said second sequence is not substantially altered in prostate cancer; and (iii) transcription associated with prostate cancer Comparing the level of the product with the level of transcript associated with normal tissue in the biological sample. A polynucleotide for quantifying transcripts associated with prostate cancer in a patient's cells, wherein the biological sample from the patient selectively hybridizes to a sequence at least 80% identical to the sequence shown in Tables 1-16 A method comprising the step of contacting with. 57. The method of claim 56, wherein the polynucleotide selectively hybridizes to a sequence that is at least 95% identical to a sequence shown in Tables 1-16. 57. The method of claim 56, wherein the biological sample is a tissue sample. 57. The method of claim 56, wherein the biological sample comprises isolated nucleic acid. 57. The method of claim 56, wherein the nucleic acid is mRNA. 60. The method of claim 59, further comprising amplifying the nucleic acid prior to the step of contacting the biological sample with the polynucleotide. 57. The method of claim 56, wherein the polynucleotide comprises a sequence shown in Tables 1-16. 57. The method of claim 56, wherein the polynucleotide is labeled. 64. The method of claim 63, wherein the label is a fluorescent label. 57. The method of claim 56, wherein the polynucleotide is immobilized on a solid surface. 57. The method of claim 56, wherein the patient is undergoing therapeutic medication management for the treatment of metastatic prostate cancer. 57. The method of claim 56, wherein the patient is suspected of having metastatic prostate cancer. A biochip comprising a plurality of polynucleotides that selectively hybridize to a sequence that is at least 80% identical to the sequences shown in Tables 1-16. i) providing a cell expressing an expression profile gene selected from the group consisting of the expression profile genes shown in Tables 1-16 or a fragment thereof;
A method of screening a drug candidate comprising: ii) adding a drug candidate to the cell; and iii) determining the effect of the drug candidate on expression of the expression profile gene. 60. The method of claim 59, wherein the determining comprises comparing the expression level in the absence of the drug candidate with the level of expression in the presence of the drug candidate.